Epitope and Paratope Mapping Reveals Temperature-Dependent Alterations in the Dengue-Antibody Interface.

Article

Epitope and Paratope Mapping Reveals Temperature-Dependent Alterations in the DengueAntibody Interface Graphical Abstract

Authors Xin-Xiang Lim, Arun Chandramohan, Xin-Ying Elisa Lim, James E. Crowe, Jr., Shee-Mei Lok, Ganesh S. Anand

Correspondence [email protected]

In Brief Temperature-dependent expansion in strains of DENV2 alters modes of antibody interaction. Lim et al. uncovered differences in modes of 2D22 interaction with DENV2 at different temperatures that are coupled to structural dynamics of DENV2. These enabled deconstruction of the heavy and light chain contributions at DENV2-2D22 interface at different temperatures.

Highlights d

Temperature-mediated DENV2 expansion alters modes of interaction of 2D22 binding

d

2D22 heavy chain interactions remain conserved across all conformations of DENV2

d

Temperature-dependent expansion of DENV2 is dampened by 2D22 complexation

Lim et al., 2017, Structure 25, 1–12 September 5, 2017 ª 2017 Elsevier Ltd. http://dx.doi.org/10.1016/j.str.2017.07.007

Please cite this article in press as: Lim et al., Epitope and Paratope Mapping Reveals Temperature-Dependent Alterations in the Dengue-Antibody Interface, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.07.007

Structure

Article Epitope and Paratope Mapping Reveals Temperature-Dependent Alterations in the Dengue-Antibody Interface Xin-Xiang Lim,1 Arun Chandramohan,1 Xin-Ying Elisa Lim,1,2,3 James E. Crowe, Jr.,4 Shee-Mei Lok,1,2,3 and Ganesh S. Anand1,5,* 1Department

of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore in Emerging Infectious Diseases, Duke-National University of Singapore Graduate Medical School, 8 College Road, Singapore 169857, Singapore 3Centre for BioImaging Sciences, CryoEM Unit, Department of Biological Sciences, National University of Singapore, Singapore 117557, Singapore 4Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Nashville, TN 37232-0417, USA 5Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2017.07.007 2Program

SUMMARY

Uncovering mechanisms of antibody-mediated neutralization for viral infections requires epitope and paratope mapping in the context of whole viral particle interactions with the antibody in solution. In this study, we use amide hydrogen/deuterium exchange mass spectrometry to describe the interface of a dengue virus-neutralizing antibody, 2D22, with its target epitope. 2D22 binds specifically to DENV2, a serotype showing strain-specific structural expansion at human host physiological temperatures of 37 C. Our results identify the heavy chain of 2D22 to be the primary determinant for binding DENV2. Temperature-mediated expansion alters the mode of interaction of 2D22 binding. Importantly, 2D22 interferes with the viral expansion process and offers a basis for its neutralization mechanism. The relative magnitude of deuterium exchange protection upon antibody binding across the various epitope loci allows a deconstruction of the antibody-viral interface in host-specific environments and offers a robust approach for targeted antibody engineering.

INTRODUCTION Dengue virus (DENV) is the causative agent for dengue fever and causes more than 400 million cases of dengue infection annually (Bhatt et al., 2013). Currently, licensed commercial vaccines show only moderate efficacy (Capeding et al., 2014; Sabchareon et al., 2012) and there are no alternative antiviral therapeutics (Bennett et al., 2010; Halstead, 2012; Holmes and Twiddy, 2003; Sabchareon et al., 2012). The antigenic diversity in viruses of the four co-circulating DENV serotypes (DENV1-4) (Guzman et al., 2010) greatly limit the development of dengue vaccines and antiviral therapeutics. Secondary infection by heterotypic dengue sero-

types has been shown to increase the risk of severe and life-threatening dengue hemorrhagic fever and dengue shock syndrome, associated with positive laboratory assays for antibody-dependent enhancement (Halstead, 2012). Therefore, effective antiviral therapeutics must be able to elicit strong neutralizing activity against all four DENV serotypes. DENV, a single-stranded RNA envelope virus, is a member of the Flaviviridae family and requires a mosquito vector (Aedes sp.) for its transmission (Westaway et al., 1985). The mature DENV particle consists of three structural proteins: capsid (C), envelope (E), and the membrane (M) protein (Lindenbach and Rice, 2001). The 180 copies of E and M protein heterodimers are arranged in icosahedral symmetry forming a herringbone pattern on the outer shell of the virus (Kuhn et al., 2002). C proteins complexed with the single-stranded plus sense RNA genome constitute the core of the virus particle and are encapsulated by the lipid bilayer membrane (Ma et al., 2004). DENV pathogenesis is initiated upon DENV transmission from a dengue-harboring mosquito to a human host during a blood meal (Westaway et al., 1985). During this transmission, temperature has been shown to trigger varying degrees of structural expansion, most prominent in certain strains of DENV2 (Fibriansah et al., 2013; Kostyuchenko et al., 2014; Zhang et al., 2013b). Following transmission, DENV particles attach onto host cell receptors and undergo clathrin-mediated endocytosis (Chu and Ng, 2004; van der Schaar et al., 2008). Fusion with the lower pH endosome triggers large-scale pH-dependent conformational transitions in the viral outer coat E protein (Klein et al., 2013; Modis et al., 2004). These changes expose the fusion loop on DENV E protein to prime association to the endosomal membrane, leading to virus and host endosomal membrane fusion (Klein et al., 2013; Modis et al., 2004). During key phases of DENV pathogenesis, the E protein undergoes large-scale conformational changes in response to host-specific environmental perturbations (Mukhopadhyay et al., 2005). The predominant role of E protein in the DENV life cycle makes this protein the major antigenic target for antibodies (Feighny et al., 1994; Roehrig, 2003). Structures of E protein in these various conditions have been solved by X-ray crystallography Structure 25, 1–12, September 5, 2017 ª 2017 Elsevier Ltd. 1


and cryoelectron microscopy (cryo-EM) (Austin et al., 2012; Cherrier et al., 2009; Cockburn et al., 2012; Dowd and Pierson, 2011; Fibriansah et al., 2015; Lok et al., 2008). These structures, however, represent only static endpoint states and offer limited predictive insights into the large-scale conformational changes that could alter the dynamic properties of epitopes presented on the surface of DENV E proteins. Mature viral particles are intrinsically metastable and consequently respond to diverse perturbations within the host such as temperature, pH, osmolality, chemical environment, etc., to steer the infectivity process. This is the least-understood step in viral infectivity as static images of viral particles offer no insights into these changes in viral particle dynamics accompanying infection of new host cells. Furthermore, how neutralizing antibodies might alter the dynamics of viral particles and their responses to perturbations unique to the host’s environment remain a mystery. This necessitates not only a map of an antibody-whole viral particle interface but also the effects of diverse host-specific perturbations. Amide hydrogen/deuterium exchange mass spectrometry (HDXMS) is a powerful method to probe dynamics (seconds and longer timescales) of intact viral particles in solution (Wang et al., 2001; Wang and Smith, 2005) at peptide resolution by measuring the increment in mass when backbone amide hydrogens exchange with solvent deuterium (Wales and Engen, 2006; Zhang and Smith, 1993). The rates of hydrogen/deuterium exchange depend upon both hydrogen bonding propensities and solvent accessibility of proteins/protein assemblies, and thereby provide a thermodynamic readout of protein interaction interfaces and protein conformation changes (Englander and Kallenbach, 1983; Woodward et al., 1982). We recently applied HDXMS to probe temperature-specific expansion of dengue and monitored changes across all structural proteins in DENV1 (PVP159) and DENV2 (NGC). Coordinated temperature-specific changes were found to be serotype-specific and were observed only in intact DENV particles (Lim et al., 2017). Human host temperatures of 37 C are critical for examining viral particle-antibody complexes as these represent the physiological condition for the immune response; and, secondly, a temperature change to 37 C/40 C represents an important host-specific stimulus that likely initiates the infectivity phase of the dengue viral particle (Lim et al., 2017). Temperaturedependent changes on whole viral particles including antibody epitope sites, would alter the stoichiometry and stability of antibody interactions. The effects of host-specific perturbations on viral-antibody interactions in the context of the intact dengue viral particle are unknown. Indeed, many flavivirus antibodies exhibit temperature-dependent binding and neutralization (Austin et al., 2012; Dowd et al., 2014; Sabo et al., 2012), and epitope accessibility is a primary factor affecting the potency of neutralizing antibodies (Dowd and Pierson, 2011). A DENV2-specific monoclonal antibody, 2D22, isolated from a patient with prior DENV2 infection (Fibriansah and Lok, 2016), exhibited differential modes of binding to the temperaturespecific expanded and unexpanded DENV2 (NGC) (Fibriansah et al., 2015). The sub-nanometer resolution (6.5 A˚) cryo-EM structures of the unexpanded DENV2 (PVP94/07 and NGC strains) bound with the Fab fragment of 2D22 at 28 C showed the epitope and paratope interaction interfaces, revealing full occupancy of all 180 epitope sites by the Fab fragment of 2 Structure 25, 1–12, September 5, 2017

2D22 (Fibriansah et al., 2015). The epitopes of 2D22 are shown to span across the protomers within the E-dimer, suggesting that the neutralization mechanism of 2D22 involves locking of E-dimers on the surface of DENV2 (Fibriansah et al., 2015). Since the NGC strain of DENV2 undergoes an expansion at the human host-specific temperature of 37 C, it is important to map the interactions of 2D22 with the expanded virus at 37 C to better understand its mechanism of neutralization, as the expanded form of the virus would be the one encountered by the host’s immune system. The cryo-EM structure of expanded DENV2 (NGC) complexed with the Fab fragment of 2D22 at 37 C, at a resolution 21 A˚, does not offer detailed insights into the epitope and paratope of the virus-antibody interface. Nevertheless, this structure revealed significant differences in valency and mode of antibody interactions in the two conditions. At 37 C, only 120 out of the 180 epitope sites on the expanded DENV2 (NGC) were occupied by the Fab fragment of 2D22 (Fibriansah et al., 2015). This temperature switch is associated with induction of major changes across the viral particle, including the 2D22 epitope region (Lim et al., 2017). This finding, together with the observed separation of the E protein dimers upon expansion of DENV2 (NGC), likely indicate that changes occur in the conformation at the 2D22 epitope in expanded DENV2 (NGC) evident from the reduced valency at the higher temperature. These observations confirm that temperature-specific conformational changes in DENV2 (NGC) prior to antibody binding are critical for antibody recognition and neutralization. Subjecting unexpanded DENV2 (NGC) pre-complexed with the Fab fragment of 2D22 to a temperature switch from 28 C to 37 C, resulted in formation of a conformationally distinct expansion intermediate (Fibriansah et al., 2015). This implied that the unique binding mode of only 120 copies of 2D22 was sufficient for effective DENV2 (NGC) neutralization (Fibriansah et al., 2015). In this study, we extended the application of HDXMS to map the epitope and paratope that formed the interaction interface, on DENV2 (NGC)-2D22 complex, in the unexpanded (UN) and temperature-expanded (EXP) states, to unravel the distinct modes of 2D22-DENV interactions and sensitivity of 2D22 binding to temperature-dependent expansion of DENV2 (NGC). The large magnitude temperature-dependent changes in the E protein from whole, intact DENV2 (NGC) viral particles characterized previously (Lim et al., 2017) represent important probes to monitor how complexation with the Fab fragment of 2D22 altered temperature expansion of DENV2 (NGC) (Figure 1). Mapping the interface of an antibody directly with its epitope on the intact infectious virus in solution, and at conditions that mimic both the infectious phase and physiologic site of immune response across the whole viral particle rather than the constituent viral proteins, represents a major advance in epitope and paratope mapping and offers closer insights into antibody action at the target viral surface. In the work described here, the designations 2D22 and DENV2 refer to the Fab fragment of 2D22 and DENV2 (NGC), respectively (Lim et al., 2017). Our results reveal that 2D22 interactions with unexpanded DENV2 particles (2D22-DENV2UN) are mediated by both the heavy and light chains, while 2D22 interactions with expanded DENV2 (2D22-DENV2EXP) are mediated solely by heavy chain interactions with E protein. These findings show that heavy chain interactions in 2D22 constitute the principal determinant for


VECTOR

Figure 1. Hydrogen/Deuterium Exchange Mass Spectrometric Analysis of 2D22DENV2 Complexes at 28 C and 37 C

HOST

(≤ 28 °C) Virus Fab 2D22 Unexpanded DENV2 (DENV2UN)

Transmission 2D22-unexpanded DENV2 (2D22- DENV2UN)

?

2D22-prebound DENV2 subjected to expansion (2D22- DENV2UN-37 °C)

(37 °C)

Fab 2D22

Expanded DENV2 (DENV2EXP)

?

2D22-expanded DENV2 (2D22-DENV2EXP)

antibody recognition and are sufficient for neutralization of DENV2. By inducing temperature-dependent expansion of the 2D22-unexpanded DENV2 complex (2D22-DENV2UN-37 C), we observed concomitant decreases in deuterium exchange at temperature-specific loci across E and M proteins of DENV2, suggesting a reduction of temperature-induced changes upon expansion of 2D22 pre-complexed DENV2. These results importantly reveal differences in the modes of antibody interaction with DENV2 at different temperatures that are coupled to structural dynamics of DENV2. These maps under different temperatures enabled deconstruction of the contributions of the heavy and light chain at the DENV2 virus-2D22 interface. The findings have enormous implications in uncovering mechanisms of antibody action under host-specific environments and provides a framework for rational antibody engineering. RESULTS Epitope and Paratope Mapping of 2D22 on Unexpanded DENV2 at 28 C We first set out to map the epitopes and paratopes for the unexpanded 2D22-DENV2UN interface at 28 C by HDXMS. Purified unexpanded DENV2 particles were equilibrated with 2D22 at 28 C at a molar ratio of one E protein: one 2D22 for 30 min and deuterium exchange was initiated by a 10-fold dilution of the Fab-virus complex in deuterated buffer. Deuterium exchange was carried out for 1 min and rapidly quenched by reducing the pHread to 2.5 followed by online pepsin proteolysis, chromatographic separation, and mass spectrometry analysis of all pepsin fragmentation peptides, as described in the STAR Methods. Fast-exchanging amides have been demonstrated to be important reporters for capturing changes at protein-protein interaction interfaces (Mandell et al., 2005) and for distinguishing protein-protein interface interactions from long-range conformational changes (Chandramohan et al., 2016). To monitor the fastexchanging amides, we carried out a time course of deuterium exchange of whole DENV2 viral particles from t = 1 to t = 60 min, published in a previous study (Lim et al., 2017). These results indicated that the relative deuterium exchange across all

Top panel: mapping the epitope and paratope of 2D22 on 2D22 unexpanded DENV2 complex (2D22-DENV2UN) at a vector temperature of 28 C by HDXMS. Bottom panel: epitope and paratope mapping of 2D22 in complex with expanded DENV2 (2D22-DENV2EXP) at 37 C by HDXMS. Monitoring temperature-dependent changes accompanying expansion of 2D22 prebound DENV2 by increasing the temperature from 28 C to 37 C (2D22-DENV2UN-37 C). All structures of unexpanded (DENV2UN), expanded DENV2 (DENV2EXP), and Fab 2D22-DENV2 complexes are represented in cartoon. E proteins constituting the 5-, 3-, and 2-fold icosahedral vertices are represented in yellow, orange, and red, respectively. Heavy and light chain of Fab 2D22 are represented in cyan and pink, respectively.

pepsin fragment peptides of E protein from DENV2, measured over a time course of t = 1, 5, 10, and 60 min, showed only small magnitude time-dependent increases in exchange after the first minute of exchange, across this time series. This suggested that 1 min of deuterium exchange represented an optimal window where deuterium exchange had occurred at a majority of fastexchanging amides in DENV2, and thus represented an optimum time point for monitoring deuterium exchange and to probe perturbational changes on whole DENV2 particles, including antibody interactions. It must be noted that the optimal timescale window for monitoring fast-exchanging amides would be protein/protein complex/viral particle-specific. Therefore, in this study, where we set out to map the epitopes and paratopes of 2D22 on DENV2 and to capture the effects of host-specific temperature perturbation on 2D22 binding, a deuterium exchange of 1 min offered an effective probe for mapping studies. Sequencing of all pepsin proteolysis peptides from DENV2 C, E, and M protein and 2D22 heavy and light chain provided sequence coverages of 10%, 77.2%, 56%, 89.8%, and 71.3%, respectively (Figure S1). Deuterium uptake for all peptides was measured as described in the STAR Methods, and the differences in deuterium exchange in peptides of DENV2 C, E, and M protein between 2D22-DENV2UN and DENV2UN at 28 C (Lim et al., 2017) were determined by comparing the average deuterium uptake in peptides across these two states (Figure 2A). The average differences in deuterium uptake for peptides across the E and M protein sequence are represented as individual difference plots (Figures 2A and S2A). Regions showing decreases in deuterium exchange between 2D22-DENV2UN and DENV2UN were observed only in peptides from E protein (57–69, 78–107, and 122–163) (Figure 2B). These regions mapped to the previously reported 2D22 heavy chain (residues 57–69 and 78– 107) and light chain (residues 122–163) epitopes (Fibriansah et al., 2015) (Figure 2C). A surface representation of the 2D22DENV2UN shows that the peptides that we have identified form two stretches of contiguous surfaces: a larger heavy chain interface and another smaller footprint contributed by the light chain (Figure S3). We did not observe differences in deuterium exchange between 2D22-DENV2UN and DENV2UN in any of the Structure 25, 1–12, September 5, 2017 3


721

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2D22-unexpanded DENV2 at 28 °C

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280 296

Deuterium Differences

L chain-virus interface

Heavy chain (H)

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Fab 2D22 heavy chain peptide

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2D22-unexpanded DENV2 at 28 °C 717

152GNDTGKHG

E-protein peptide

Unexpanded DENV2 at 28 °C


CKHSMVDRG WGNGCGL107 (m/z=2865.3)

B

Deuterium exchange t=1 min

Intensity units

83NEEQDKRFV

Undeuterated

21-30

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Fab 2D22 light chain peptide

Figure 2. Epitope and Paratope Mapping of 2D22 Unexpanded DENV2 Complex (2D22-DENV2UN) at 28 C by HDXMS (A) Isotopic mass envelope of representative E protein peptides that spanned the heavy and light chain epitopes from DENV2UN (black spectra) and 2D22DENV2UN (purple spectra) at 28 C after 0 and 1 min of deuterium exchange. Amino acids of peptides spanning the heavy and light chain are indicated as cyan and pink letters, respectively. Red dashed lines represent the average centroid of the mass envelope. (B) Differences in deuterium exchange in pepsin-proteolyzed peptides of DENV2 E protein between free DENV2UN and 2D22-DENV2UN after 1 min of deuterium exchange at 28 C are represented in a difference plot. For the x axis, each node represents a pepsin-proteolyzed peptide, and all peptides are listed from the N to C terminus. The y axis shows differences in number of deuterons between the two states compared. Domain organization of DENV2 E protein is indicated below the difference plot. (C) Deuterium exchange differences in E protein peptides between free DENV2UN and 2D22-DENV2UN are mapped onto the cryo-EM structure of an E protein dimer (PDB: 4UIF) from the DENV2UN. The E-dimer is represented as a ribbon and one of the protomers is shaded in light gray. The three E protein ectodomains are labeled DI, DII, and DIII. (D and E) The difference plot of the heavy chain that interacts with DENV2UN (D) and the difference plot of the light chain that interacts with DENV2UN (E). Differences in deuterium exchange in pepsin-proteolyzed peptides of 2D22 heavy and light chains after 1 min of deuterium exchange between free 2D22 and 2D22-DENV2UN at 28 C are represented in the difference plots. Peptides exhibiting decreased deuterium differences that spanned residues reported to be the 2D22 epitope (E protein) and the paratope (Fab 2D22) by a previous cryo-EM study (Fibriansah et al., 2015) are highlighted in regions of blue and pink, respectively. Differences in deuterium exchange lower than 0.5 D (greater than 0.5 D in magnitude) were considered significant (red dashed lines) across the two states compared. SE values for each peptide are shown as purple shaded regions along the x axis in the difference plots and represent the sum of the single sigma standard deviations of each of the two conditions being compared. (F) Deuterium exchange differences between free 2D22 and 2D22-DENV2UN in the peptides of 2D22 heavy (H) and light (L) chains are mapped onto the cryo-EM structure of 2D22 (PDB: 4UIF). The 2D22 epitope and paratope reported in a previous cryo-EM study are indicated as spheres. Epitope and paratope residues of the heavy chain are indicated with a cyan circle. Epitope and paratope residues of the light chain are indicated with a pink circle. Regions with no peptide coverage are in gray.

peptides from M protein (Figure S2A). This finding is consistent with the previous study showing that the surface epitopes of 2D22 on DENV2 map solely onto the E protein (Fibriansah et al., 2015). Next, we examined the paratope of 2D22 by mapping deuterium exchange differences in peptides of the heavy and light chain between 2D22-DENV2UN at 28 C and the unbound 2D22. Peptides showing significant decreases were localized to regions spanning residues 46–59, 67–83, and 94–106 in the 2D22 heavy chain (Figure 2D) and residues 29–74 in the light chain (Figure 2E). These regions are consistent with the previously reported paratope of 2D22 (Fibriansah et al., 2015), and, 4 Structure 25, 1–12, September 5, 2017

among these, peptides 46–59 from the heavy chain and 48–74 from the light chain displayed the greatest magnitude decreases in deuterium exchange (Figure 2F). Capturing the 2D22 Footprint and Conformational Changes on Expanded DENV2 at 37 C Although we mapped the epitopes and paratopes in 2D22DENV2UN at 28 C, DENV2UN may not represent the conformation that antibodies encounter in the human host during an infection, as DENV2 undergoes temperature-dependent expansion at the host temperature of 37 C (Fibriansah et al., 2013; Lim et al., 2017). This expansion revealed previously cryptic epitopes (Lok


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Deuterium Differences

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H chain-virus interface 1

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KEIK163 (m/z=3441.5)

E-protein peptide


717

152GNDTGKHG

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Intensity units

83NEEQDKRFV

Undeuterated

60-66

A


Figure 3. Expansion of DENV2 under a Human Host Temperature of 37 C Affects 2D22 Binding (A) Isotopic mass envelope of representative E protein peptides that spanned the heavy and light chain epitopes from expanded DENV2 (DENV2EXP) (black spectra) and 2D22-expanded DENV2 complex (2D22-DENV2EXP) (green spectra) at 37 C after 0 and 1 min of deuterium exchange. Amino acid sequences of peptides spanning the heavy and light chain are indicated in cyan and pink letters, respectively. The red dashed line indicates the average centroid of the mass envelope. (B) Differences in deuterium exchange in pepsin-proteolyzed peptides of DENV2 E protein after 1 min of deuterium exchange between free DENV2EXP and 2D22DENV2EXP at 37 C are represented in a difference plot. For the x axis, each point represents a pepsin-proteolyzed peptide, and all peptides are listed from the N to the C terminus. The y axis shows differences in the number of deuterons between the two states compared. Domain organization of DENV2 E protein is indicated below the difference plot. (C–E) Differences in deuterium uptake after 1 min of deuterium exchange between free DENV2EXP and 2D22-DENV2EXP at 37 C are mapped onto an E-dimer from the cryo-EM structure of expanded DENV2 (PDB: 3ZKO). The three E protein ectodomains are labeled DI, DII, and DIII. Difference plots representing the differences in deuterium exchange in pepsin-proteolyzed peptides of 2D22 (D) heavy and (E) light chain after 1 min of deuterium exchange between free 2D22 and 2D22-DENV2EXP at 37 C. Peptides exhibiting decreased deuterium differences that spanned residues reported to be 2D22 epitope (E protein) and paratope (Fab 2D22) by a previous cryo-EM study (Fibriansah et al., 2015) are indicated in blue and pink, respectively. Differences in deuterium exchange lower than 0.5 D (greater than 0.5 D in magnitude) were considered significant (red dashed lines) across the two states compared. SE for each peptide is represented as green shaded region along the x axis. (F) Deuterium exchange differences in 2D22 heavy (H) and light (L) chain peptides between free Fab 2D22 and 2D22-DENV2EXP are mapped onto the cryo-EM structure of 2D22 (PDB: 4UIF). Regions with no peptide coverage are in gray. 2D22 epitope and paratope residues reported by cryo-EM are represented as spheres. Residues constituting the reciprocal epitope and paratope interaction interface mediated by the heavy chain and light chain of 2D22 are circled cyan and pink, respectively.

et al., 2008) and altered conformation of surface exposed epitopes that may affect antibody binding or its interaction surface. Therefore we further investigated the binding of Fab 2D22 to the expanded DENV2 (DENV2EXP) at 37 C. Equilibrated DENV2 at 37 C was incubated with 2D22 for 30 min in an equilmolar ratio of E protein and 2D22 and subjected to 1 min of deuterium exchange. It should be noted that the intrinsic rate of hydrogen/deuterium exchange is dependent on temperature (Bai et al., 1993). Previously, we estimated a 2.33-fold increase in the intrinsic rate of HDX at 37 C compared with that at 28 C (Lim et al., 2017). This 2.33-fold increase in HDX rate only accounts for a relatively minor contribution to the measured differences in deuterium exchange between the two temperatures at 1 min of deuterium exchange compared with the changes in protection factors

upon binding of 2D22 with DENV2. Hence, the differences in deuterium exchange between the 2D22-DENV2 complex and free DENV2 at the respective temperatures describe solely effects of 2D22 binding and any associated conformational changes. Differences in deuterium exchange were determined by comparing the average differences in deuterium uptake of pepsin-proteolyzed E protein peptides between the 2D22expanded DENV2 complex (2D22-DENV2EXP) and DENV2EXP at 37 C (Lim et al., 2017) (Figure 3A), and are represented in a difference plot (Figure 3B). At 37 C, E protein peptides spanning residues 57–69 and 78–107 showed significant decreases in deuterium exchange upon 2D22 binding. These regions corresponded to the heavy chain binding epitopes observed at 28 C (Fibriansah et al., 2015), and the differences in exchange Structure 25, 1–12, September 5, 2017 5


indicate heavy chain binding to the E protein surface. In addition, we observed decreases in deuterium exchange in E protein peptides spanning residues 238–260. This locus was observed previously to display the greatest temperature-specific changes upon DENV2 expansion, indicating that binding of the heavy chain reduced temperature-specific changes at this region (Figure 3C). Consistently, concomitant decreases in deuterium exchange were also observed in a peptide spanning the stem region of M protein (27–45) (Figure S2B), highlighting the cooperativity of temperature-dependent changes across structural proteins in DENV2. Interestingly, we did not observe a decrease in deuterium exchange in the light chain epitopes (152–163). This finding revealed that the light chain did not contribute significantly to the interface in the expanded conformation of DENV2 at 37 C (Figure 3B). This was confirmed further by close examination of the paratope interface by monitoring the differences in deuterium uptake in peptides across the heavy and light chain of 2D22 upon binding with DENV2EXP. The average deuterium differences in pepsin-proteolyzed peptides of the 2D22 heavy and light chain between 2D22-DENV2EXP and unbound 2D22 at 37 C are represented in a difference plot (Figures 3D and 3E). Decreases in deuterium uptake were observed only in peptides spanning the 2D22 heavy chain paratopes (46–59, 67–83, and 94–106) (Figure 3D). Consistently, we did not detect significant differences in deuterium uptake for any 2D22 light chain peptides (Figure 3E). This finding correlated perfectly with the absence of deuterium exchange differences on the light chain epitopes on E protein (Figures 3E and 3F). This result indicates the absence of interactions between the 2D22 light chain and E protein on expanded DENV2. Together, these experiments revealed a conformation-dependent binding mode and mechanism of neutralization of DENV2 by 2D22 (Figures 3D and 3E). Through parallel deuterium exchange analysis of the peptides of 2D22 and DENV structural proteins in the 2D22-DENV2 complexes at the two temperatures, the results therefore describe a predominantly heavy chain-mediated binding and neutralization of the DENV2 virus by 2D22 at 37 C. Binding of 2D22 to DENV2 Dampens ExpansionAssociated Temperature-Dependent Changes We next sought to examine the role of the 2D22 light chain in binding to DENV2 formed at 28 C. DENV2UN was equilibrated with 2D22 at a ratio of one E protein: one 2D22 Fab for 30 min at 28 C, and expansion of the 2D22-DENV2UN was initiated by incubation at 37 C for 30 min (2D22-DENV2UN-37 C). The 2D22-DENV2UN-37 C was subjected to 1 min of deuterium exchange and the deuterium uptake for peptides of DENV2 E protein; the heavy and light chains of 2D22 were measured similarly. Differences in deuterium uptake across peptides of the E protein (Figures 4A and 4B) and 2D22 (Figures 4C and 4D) were determined by comparing the deuterium exchange between 2D22DENV2UN-37 C with DENV2EXP and unbound 2D22 at 37 C, respectively. Decreases in deuterium exchange were observed in peptides spanning both the heavy chain epitope (residues 57–69 and 78–107) on E protein (Figures 4E and 4F) and the reciprocal heavy chain paratope (residues 46–59, 67–83, and 94–106) on 2D22 (Figure 4G). In addition, concomitant 6 Structure 25, 1–12, September 5, 2017

decreases in deuterium exchange also were observed in peptides that spanned the light chain epitope (152–163) on E protein and the reciprocal light chain paratope (48–74) on 2D22 (Figures 4E and 4G). Notably, both 2D22 binding and dampened expansion of DENV2 were detected by decreases in deuterium exchange. We could distinguish between 2D22 binding and reduced expansion by comparing the loci displaying decreased deuterium exchange with the epitope footprints of 2D22 on DENV2 at 28 C and 37 C captured by HDXMS. We reasoned that any differences in deuterium exchange in peptides of the heavy and light chain epitopes on E protein can be attributed to complexation alone, if a corresponding decrease in deuterium exchange is observed in peptides spanning the reciprocal paratope, in the respective heavy and light chains of 2D22. Therefore, we have confirmed that the above-described loci showing decrease in deuterium exchange at the epitopes are primarily due to 2D22mediated complexation. Apart from peptides spanning the 2D22 epitopes on E protein, decreases in deuterium exchange also were observed at previously characterized temperature expansion loci on E protein such as the intradimeric interface (238–260), stem (431–448), and transmembrane helices (465–486) (Figures 4B, 4E, and 4F), which are not involved in direct interactions with 2D22 (Lim et al., 2017). These findings indicated that temperaturedependent conformational changes in 2D22-DENV2UN-37 C were altered by 2D22 binding. Moreover, concomitant decreases in deuterium exchange observed in peptides spanning the previously characterized temperature expansion loci on M protein in DENV2 suggest that 2D22 dampened temperaturedependent expansion of 2D22-DENV2UN-37 C (Figure S2C) (Lim et al., 2017). Collectively, the epitope footprints captured as differences in deuterium exchange at characteristic regions of the E protein from these three different temperature conditions revealed that light chain interactions dampened temperaturedependent expansion of DENV2 when they are established prior to treatment with higher temperatures. Comparison of loci exhibiting decreased deuterium exchange at characteristic regions of the E protein in three different conditions (1) 2D22-DENV2UN at 28 C, (2) 2D22-DENV2EXP at 37 C, and (3) 2D22-DENV2UN-37 C (Figures 2, 3, and 4, respectively) provide broader insights into the mechanism of binding and neutralization of DENV2 by 2D22. The observation that 2D22 heavy chain interactions were preserved under all three temperature conditions, whereas light chain interactions were detected only when 2D22 binding preceded expansion, indicated that 2D22 heavy chain interactions represent the most important determinants for binding at the two temperatures leading to virus neutralization. DISCUSSION Temperature Alters Mode of 2D22 Interactions with DENV2 The life cycle of DENV necessitates that the surface E proteins undergo a cascade of conformational changes triggered by diverse environmental cues including pH and temperature, or interactions with other biological molecules such as host proteins that serve as receptors (Mukhopadhyay et al., 2005), leading to


719

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78-107 122-154 152-163 176-193

31-42

Legend 2D22-unexpanded DENV2 subjected to 37 °C

-2

DENV2 temperaturedependent changes

H

Legend Deuterium Differences

0.5 Da cut-off

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152GNDTGKHG

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83NEEQDKRFV

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94-104

67-83

60-66

46-59

D < -0.5

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4-10 19-30

D < -1


Figure 4. 2D22 Binding to DENV2 Limits Temperature-Dependent Expansion (A) Isotopic mass envelope of representative E protein peptides that spanned the heavy and light chain epitopes from expanded DENV2 (DENV2EXP) (black spectra) at 37 C and 2D22-prebound DENV2 subjected to expansion (2D22-DENV2UN-37 C) (orange spectra) at 37 C after 0 and 1 min of deuterium exchange. Amino acid sequences of peptides spanning the heavy and light chain are indicated as cyan and pink letters, respectively. The red dashed lines indicate the average centroid of the mass envelope. (B–D) Differences in deuterium exchange in pepsin-proteolyzed peptides of DENV2 E protein after 1 min of deuterium exchange between free DENV2EXP and 2D22-DENV2UN-37 C are represented in a difference plot. For the x axis, each point represents a pepsin-proteolyzed peptide, and all peptides are listed from the N to the C terminus. The y axis shows differences in the number of deuterons between the two states compared. The black dashed lines represent absolute differences in deuterium exchange across all the peptides from the E protein between 28 C and 37 C mapped from a previous HDXMS study (Lim et al., 2017). These temperature-dependent differences in deuterium exchange were determined by subtracting the deuterium uptake of the peptide at 28 C from that at 37 C. Hence, previously mapped temperature-dependent changes were represented as negative magnitude of deuterium-exchange differences. Domain organization of the DENV2 E protein is indicated below the difference plot. Differences in deuterium exchange in pepsin-proteolyzed peptides of 2D22 (C) heavy and (D) light chain after 1 min of deuterium exchange between free 2D22 and 2D22-DENV2UN-37 C are represented in difference plots. Peptides exhibiting decreased deuterium differences that spanned residues reported by a previous cryo-EM study (Fibriansah et al., 2015) to be 2D22 epitope (E protein) and paratope (2D22) are highlighted in regions of blue and pink, respectively. Differences in deuterium exchange lower than 0.5 D (greater than 0.5 D in magnitude) were considered significant (red dashed line) between the two states compared. SE values for each peptide are represented as orange shaded regions along the x axis. (E and F) Differences in deuterium exchange at 1 min are mapped onto the structure of E dimers from the cryo-EM structure of 2D22-DENV2UN-37 C intermediate (PDB: 4UIH) and displayed in orthogonal views. The three E protein ectodomains, stem helices, and transmembrane helices are labeled DI, DII, DIII, S, and TM, respectively. (G) Deuterium exchange differences in 2D22 heavy (H) and light (L) chain peptides between free 2D22 and 2D22-DENV2UN-37 C are mapped onto the cryo-EM structure of 2D22 (PDB: 4UIF). Regions with no peptide coverage are in gray. 2D22 epitope and paratope residues reported by a previous cryo-EM study (Fibriansah et al., 2015) are represented as spheres. Residues constituting the reciprocal epitope and paratope interaction interface mediated by the heavy chain and light chain of 2D22 are circled cyan and pink, respectively.

disassembly of the viral coat and release of the nucleic acid core into the host cell. Disassembly of viral particles at host target sites are thereby critical for dengue viral infectivity. These coordinated conformational changes alter dynamic properties and epitope accessibility on DENV E protein, which in turn potentially impact binding and potency of neutralizing antibodies (Austin et al., 2012; Sukupolvi-Petty et al., 2013). Some strains of DENV2 have been shown to undergo temperature-dependent expansion during vector (28 C) to host (37 C) transmission (Fibriansah et al., 2013; Zhang et al., 2013b), and we have observed previously using HDXMS that the intradimeric E protein interface showed the greatest temperature-specific change upon DENV2

expansion (Lim et al., 2017). In this study, we applied HDXMS to map the epitope, which spanned across the E protein intradimeric interface, and the paratope of the Fab fragment of 2D22 on DENV2 under host-specific temperatures. HDXMS also revealed that the heavy chain contributed a larger footprint to the 2D22-DENV2 interface relative to the light chain (Figure S3). Consequently, only the heavy chain’s interactions are retained over the light chain when the antibody is allowed to interact with pre-expanded DENV2. Through these we observed that the binding of the heavy chain remained conserved independent of the temperatures tested, whereas binding by the light chain was observed only when 2D22 binding preceded DENV2 Structure 25, 1–12, September 5, 2017 7


Figure 5. Proposed Mechanism of 2D22 Action on Unexpanded (DENV2UN) and Expanded DENV2 (DENV2EXP) Residues constituting the 2D22 heavy and light chain epitopes are represented on structures of E-dimers from DENV2UN (A) surface and (B) side view and DENV2EXP (C) surface and (D) side view at 28 C and 37 C, respectively. One E protein protomer is represented in light blue and the other in red. Loci of heavy and light chain epitopes are indicated by cyan and pink circles, respectively. Specific residues constituting the heavy and light chain epitopes are indicated and represented as spheres. The measured distances between heavy and light chain residues are indicated and represented with black dashed lines.

expansion. This is an important breakthrough where the interaction of the antibody has been mapped at peptide resolution in the context of quaternary assembly of constituent E proteins on the virus particle, and where the contributions of the heavy and light chains have been delineated. To understand the possible reasons for abolishment of light chain binding on expanded DENV2, we examined the approximate distances and relative orientations of the heavy chain epitopes (T66 from b strand and G104 from fusion loop in E protein domain II), with respect to the light chain epitopes (D154 from the glycan loop) on the cryo-EM structures of the DENV2UN (3.2 A˚) (Figures 5A and 5B) and DENV2EXP (13 A˚) (Figures 5C and 5D) prior to 2D22 binding. These residues were selected on the basis that peptides spanning these residues were identified from HDXMS data as the epitope regions in 2D22-DENV2 complexes, and are also consistent with the epitopes reported in the previous cryo-EM study (Fibriansah et al., 2015). The distances between T66 and G104 from the heavy chain and D154 from the light chain epitopes across the E-dimer in the DENV2UN are approximately 28 and 10 A˚, respectively (Figures 5A and 5B). However, distances between T66 and G104 from the heavy chain and D154 from the light chain epitopes across the E-dimer on the DENV2EXP were greater, by approximately 35 and 21 A˚, respectively (Figures 5C and 5D). It should be noted that the distances between the heavy chain and light chain epitopes on the DENV2UN represent a more accurate measurement due to the higher-resolution of the cryo-EM structure of DENV2UN (Zhang et al., 2013a) as compared with the lower-resolution cryo-EM structure of the DENV2EXP (Fibriansah et al., 2013). These distances therefore were inferred only as an estimate of the relative distances between the epitope sites. In addition to the greater distances between the heavy and light chain residues, the orientation of the epitopes on the E protein dimer also differed with the heavy chain epitope residue G104 (fusion loop) moving away 8 Structure 25, 1–12, September 5, 2017

from the light chain epitope residue D154 on the adjacent E-protomer and toward the viral membrane (Figures 5B and 5D) upon DENV2 expansion. This finding suggested that the spatial separation and altered conformation of the heavy and light chain epitopes upon DENV2 expansion, likely resulted in the abolishment of 2D22 light chain binding. Therefore, the heavy chain interactions are the principal determinants of temperature-independent binding of 2D22 to DENV2. 2D22 Binding Interferes with Viral Disassembly The immunoglobulin G form of 2D22 has been demonstrated to bind to and potently neutralize both temperature-dependent expanding and non-expanding strains of DENV2 (Fibriansah et al., 2015). Thus, 2D22-DENV2 interactions are inherently plastic as they can adapt to alternate conformations of the epitope presented on the virus surface, and both 2D22-DENV2UN and 2D22-DENV2EXP represent modes of binding associated with neutralization. Therefore, we propose that the neutralization mechanisms of mAb 2D22 depend largely on heavy chain interactions with domain II of E protein for strains of DENV2 that undergo temperature expansion, whereas neutralization of the non-expanding strains of DENV2 involves additional reduction in viral coat dynamics modulated by the binding of the heavy and light chain across unseparated E-dimers. This binding mode may possibly prevent the dissociation of E-dimers into monomers thus disrupting the low-pH E protein transitions to trimer conformation. The important class of highly potent and broadly neutralizing E-dimer-dependent epitope (EDE)-recognizing antibodies shares largely overlapping epitopes with 2D22 (Dejnirattisai et al., 2015; Rouvinski et al., 2015). These epitopes are highly conserved among the four DENV serotypes, and are mainly clustered around the b strand (67–74), fusion loop (97–106), and ij loop (246–249) in DII from one E-monomer, and the 150 loop (148–159) from the


VECTOR A

HOST

(≤ 28 °C) B

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Fab 2D22 Unexpanded U d d DENV2 (DENV2UN)

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2D22-prebound DENV2 subjected to expansion (2D22- DENV2UN-37 °C)

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Fab 2D22 Legend

Expanded DENV2 (DENV2EXP)

2D22-expanded DENV2 (2D22-DENV2EXP)

Deuterium Differences D < -1 D < -0.5 D > -0.5 No coverage

Figure 6. Host-Specific Temperature Perturbation Alters Mode of 2D22 Binding to DENV2 Top panel: cryo-EM structures of (A) DENV2UN (PDB: 3J27) and (B) 2D22-DENV2UN (PDB: 4UIF). Deuterium exchange difference across all E protein peptides after 1 min of deuterium exchange at 28 C between free DENV2UN and 2D22-DENV2UN is mapped onto the structure of 2D22-DENV2UN. Bottom panel: cryo-EM structures of (C and D) DENV2EXP (PDB: 3ZKO) and (E) expansion intermediate of 2D22-DENV2EXP-37 C (PDB: 4UIH). Deuterium exchange differences across all E protein peptides after 1 min of deuterium exchange at 37 C between DENV2EXP and 2D22-DENV2EXP, 2D22-DENV2UN-37 C are mapped onto the structures of (D) DENV2EXP (PDB: 3ZKO) and (E) expansion intermediate 2D22-DENV2EXP-37 C (PDB: 4UIH), respectively. Fab 2D22 is represented in cartoon with the heavy and light chains colored cyan and pink, respectively.

adjacent E protein subunit (Dejnirattisai et al., 2015; Rouvinski et al., 2015). We previously observed temperature-dependent changes in these loci accompanying DENV2 expansion (Lim et al., 2017). In addition, these epitopes become spatially separated with the dissociation of E-dimers upon DENV2 expansion. However, these temperature-dependent changes are serotypeand strain-specific and are not observed in DENV1 at 37 C (Lim et al., 2017). Nevertheless, at 37 C EDE antibodies bind and neutralize not only all the four serotypes of DENV but also the related flavivirus, Zika (Barba-Spaeth et al., 2016; Dejnirattisai et al., 2016). This observation highlighted the fact that EDE antibodies, such as 2D22, recognize multiple conformations of the epitope presented by the different DENV serotypes and under diverse host-specific environments. Epitope and Paratope Mapping under Host-Specific Perturbations Highlights the Potential for Rational Antibody Engineering Epitope and paratope mapping in host-like environments offer the obvious advantage of identifying epitopes that are critical for antibody binding, mimicking conditions closer to when the virus encounters the host’s immune system. Structural elucidation of antibody-virus complexes by X-ray crystallography and cryo-EM provide high-resolution insights into the interactions between epitope and paratope residues (reviewed in Lok, 2016). These structures represent stable endpoint states and are limited in offering predictive insights into how large-scale conformational transitions in viral coat proteins that are specific

to host environment, affect antibody interactions. HDXMS offers orthogonal advantages to map epitopes and paratopes based on a thermodynamic readout of antibody-virus complexes under host-specific conditions at peptide resolution. In addition, through the use of additional structural viral capsid proteins not on the surface as conformational probes, it offers a comprehensive readout of the long-range conformational effects of antibody binding on virus architecture. The high-resolution insights of all stable residual contacts by X-ray crystallography and cryo-EM, combined together with HDXMS-based mapping of effects of unique host-specific conditions of temperature, osmolality, or pH, on the conformational dynamics of antibody-virus complexes present a powerful strategy for generating and validating optimized antibodies for antiviral therapeutic design. In this study, epitope and paratope maps of 2D22-DENV2 complexes show temperature-specific alterations in conformations (Figures 6A–6E) and highlight a heavy chain-mediated mode of binding of 2D22 at higher temperature (Figures 6C, 6D, and 6E). Interestingly, the epitope map of 2D22-DENV2UN at 28 C was captured in peptides spanning only a subset of residues reported to be part of the interface in the cryo-EM structure of the complex (Fibriansah et al., 2015). Peptides spanning the residues of the heavy chain epitopes and paratopes corresponded very well with interface residues identified by cryo-EM (Fibriansah et al., 2015). However, this correlation was weak for residues of the light chain epitope and paratope. Our findings revealed that the heavy chain interactions are primary determinants for stable binding of 2D22 to DENV2, and Structure 25, 1–12, September 5, 2017 9


consequently this interaction is maintained independent of the temperature-dependent expansion of DENV2. This underscores the importance of capturing thermodynamic differences of the virus under host-specific environments to complement the high-resolution structural snapshots obtained by X-ray crystallography and cryo-EM. Collectively, findings from these two approaches may provide insights for rational optimization in antibody engineering. An ideal DENV therapeutic antibody requires strong binding and potent neutralization against all four heterologous DENV serotypes. This strategy provides a powerful framework for capturing loci that are critical for mediating antibody interactions across the four DENV serotypes and are especially relevant for broadly neutralizing antibodies such as the important class of EDE antibodies. In this regard, antibodies such as 2D22 that are effective in holding onto antigenic loci on viral surfaces, even though the viral particle is intrinsically highly dynamic and sensitive to host-specific perturbations, are more desirable therapeutic antibodies. HDXMS on viral-antibody complexes under different host-specific environments offers a readout into the versatility of such broad-specificity antibodies in mediating specific interactions across sequence- and conformation-specific serotype differences and under changing host-specific environments. Our strategy using HDXMS for epitope and paratope mapping in antibody-whole viral particle complexes offers direct insights into the desirable features of antibodies that are able to maintain interactions leading to viral neutralization. We believe that broad flaviviral specificity antibodies would bind targets under varied host environments through alterations in both viral and viral-antibody complex dynamics. This would contribute immensely to improvements in antibody design. Effective vaccine targets must possess both high antigenicity and immunogenicity. Insights from antibody footprints captured by structural methods and HDXMS provide insights into the antigenicity, but not immunogenicity, of the epitope regions. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d d

d

KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING EXPERIMENTAL MODEL AND SUBJECT DETAILS B Purification of DENV2 NGC METHOD DETAILS B Temperature Perturbations of 2D22-DENV2 B HDXMS of 2D22-DENV2 Complexes and Fab 2D22 B Quenching HDX and Phospholipid Removal B LC/MS of 2D22-DENV2 Complexes and Fab 2D22 QUANTIFICATION AND STATISTICAL ANALYSIS B Pepsin Fragment Peptide Identification B Deuterium Uptake Measurements in Peptides B Determination of Deuterium Exchange Differences

SUPPLEMENTAL INFORMATION Supplemental Information includes three figures and can be found with this article online at http://dx.doi.org/10.1016/j.str.2017.07.007. AUTHOR CONTRIBUTIONS G.A. and X.-X.L. designed the experiments. A.C., G.A., and X.-X.L. analyzed and interpreted the results. X.-X.L. performed the HDXMS experiments. G.A. and X.-X.L. wrote the manuscript. A.C. edited the manuscript. X.Y.E.L. and S.-M.L. provided virus samples. J.E.C. provided the 2D22 Fab fragments. A.C., G.A., J.E.C., X.-X.L., and S.-M.L. contributed to manuscript revision. ACKNOWLEDGMENTS Structural mass spectrometry was carried out at the Protein and Proteomics Center (PPC), Department of Biological Sciences, NUS. This work was supported by a grant from Singapore Ministry of Education Academic research fund, Tier 3 (MOE2012-T3-1-008). This work was supported by a grant from Singapore Ministry of Education Tier 3 grant, Singapore, awarded to G.A. and S.-M.L. The National Research Foundation Investigatorship award (NRF-NRFI2016-01) and the Duke-NUS Signature Research Programme funded by the Ministry of Health -Singapore was awarded to S.-M.L. Received: April 25, 2017 Revised: July 13, 2017 Accepted: July 13, 2017 Published: August 17, 2017 REFERENCES Austin, S.K., Dowd, K.A., Shrestha, B., Nelson, C.A., Edeling, M.A., Johnson, S., Pierson, T.C., Diamond, M.S., and Fremont, D.H. (2012). Structural basis of differential neutralization of DENV-1 genotypes by an antibody that recognizes a cryptic epitope. PLoS Pathog. 8, e1002930. Bai, Y., Milne, J.S., Mayne, L., and Englander, S.W. (1993). Primary structure effects on peptide group hydrogen exchange. Proteins 17, 75–86. Barba-Spaeth, G., Dejnirattisai, W., Rouvinski, A., Vaney, M.C., Medits, I., Sharma, A., Simon-Loriere, E., Sakuntabhai, A., Cao-Lormeau, V.M., Haouz, A., et al. (2016). Structural basis of potent Zika-dengue virus antibody crossneutralization. Nature 536, 48–53. Bennett, S.N., Drummond, A.J., Kapan, D.D., Suchard, M.A., Munoz-Jordan, J.L., Pybus, O.G., Holmes, E.C., and Gubler, D.J. (2010). Epidemic dynamics revealed in dengue evolution. Mol. Biol. Evol. 27, 811–818. Bhatt, S., Gething, P.W., Brady, O.J., Messina, J.P., Farlow, A.W., Moyes, C.L., Drake, J.M., Brownstein, J.S., Hoen, A.G., Sankoh, O., et al. (2013). The global distribution and burden of dengue. Nature 496, 504–507. Capeding, M.R., Tran, N.H., Hadinegoro, S.R.S., Ismail, H.I.H.J.M., Chotpitayasunondh, T., Chua, M.N., Luong, C.Q., Rusmil, K., Wirawan, D.N., Nallusamy, R., et al. (2014). Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomised, observer-masked, placebo-controlled trial. Lancet 384, 1358–1365. Chandramohan, A., Krishnamurthy, S., Larsson, A., Nordlund, P., Jansson, A., and Anand, G.S. (2016). Predicting allosteric effects from orthosteric binding in Hsp90-ligand interactions: implications for fragment-based drug design. PLoS Comput. Biol. 12, e1004840. Cherrier, M.V., Kaufmann, B., Nybakken, G.E., Lok, S.M., Warren, J.T., Chen, B.R., Nelson, C.A., Kostyuchenko, V.A., Holdaway, H.A., Chipman, P.R., et al. (2009). Structural basis for the preferential recognition of immature flaviviruses by a fusion-loop antibody. EMBO J. 28, 3269–3276.

DATA AVAILABILITY

Chu, J.J., and Ng, M.L. (2004). Infectious entry of West Nile virus occurs through a clathrin-mediated endocytic pathway. J. Virol. 78, 10543–10555.

The datasets generated and/or analyzed during the current study are available from the corresponding author upon request.

Cockburn, J.J., Navarro Sanchez, M.E., Goncalvez, A.P., Zaitseva, E., Stura, E.A., Kikuti, C.M., Duquerroy, S., Dussart, P., Chernomordik, L.V., Lai, C.J.,

10 Structure 25, 1–12, September 5, 2017


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12 Structure 25, 1–12, September 5, 2017


STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

de Alwis et al., 2012

N/A

The laboratory of Michael Rossmann

N/A

Antibodies Fab fragment of 2D22 Bacterial and Virus Strains DENV2 New Guinea-C strain (NGC) Chemicals, Peptides, and Recombinant Proteins RPMI (Roswell Park Memorial Institute)

Sigma-Aldrich

R8758

HEPES (Hyclone)

GE Healthcare Life Sciences

SH30237.01

FCS (fetal calf serum)

ThermoFisher Scientific

10438018

polyethylene glycol (PEG 8000)

Sigma-Aldrich

25322-68-3

TrisHCl

Sigma-Aldrich

185-53-1

NaCl

Sigma-Aldrich

7647-14-5

EDTA

Sigma-Aldrich

60-00-4

bovine serum albumin (BSA)

Sigma-Aldrich

9048-46-8

D2O

Cambridge Isotope Laboratory Inc.

7789-20-0

NaOH

Sigma-Aldrich

1310-73-2

GnHCl

Sigma-Aldrich

50-01-1

Tris(2-carboxyethyl) phosphine-hydrochloride (TCEP-HCl)

Sigma-Aldrich

51805-45-9

titanium dioxide (TiO2)

Sigma-Aldrich

13463-67-7

Sigma-Aldrich

89051705; RRID: CVCL_Z230

Experimental Models: Cell Lines C6/36 Aedes albopictus mosquito cells Software and Algorithms PROTEIN LYNX GLOBAL SERVER version 3.0

Waters

N/A

DYNAMX Ver. 2.0 software

Waters

N/A

CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for resources and reagents should be directed to and will be fulfilled by the corresponding author, Ganesh S. Anand ([email protected]) EXPERIMENTAL MODEL AND SUBJECT DETAILS Purification of DENV2 NGC DENV2 New Guinea-C strain (NGC) stock was a kind gift from Michael Rossmann (Purdue). Suspensions of DENV2 (NGC) were produced and purified as described previously (Fibriansah et al., 2013; Kuhn et al., 2002). Briefly, C6/36 Aedes albopictus mosquito cells were cultured in RPMI (Roswell Park Memorial Institute) medium containing 25 mM HEPES and 10% FCS (fetal calf serum). The cells were grown to 80% confluency and inoculated with DENV2 (NGC) strain at multiplicity of infection of 0.1 for 2 h at 28 C. The inoculum was replaced with fresh RPMI medium containing 2% FCS and incubated for 4 days. DENV2 NGC particles were harvested from the culture medium by centrifugation and precipitation with 8% polyethylene glycol (PEG 8000) in NTE buffer (12 mM TrisHCl, pH 8.0, 120 mM NaCl and 1 mM EDTA). The virus particles were resuspended in NTE buffer and purified through 24% sucrose cushion followed by a 10% to 30% potassium tartrate gradient centrifugation. The virus band was extracted and buffer exchanged into NTE buffer. DENV2 NGC in NTE buffer was concentrated with Amicon Ultra-4 (100 kDa molecular weight cut-off) to a final volume of approximately 100 to 150 mL. The amount of virus used for HDXMS analysis was measured using the concentration of the E-protein in the DENV2 (NGC) sample and was estimated by comparing the intensity of the E-protein band with the band intensity of bovine serum albumin (BSA) standards with known concentration on an SDS-PAGE gel stained by Coomassie Blue. Concentration of DENV2 (NGC) used for the HDXMS analysis (0.25 mg/mL) hereafter refer to the amount of E-protein in the viral samples. The sample contained mostly mature DENV2 particles with only minimal immature virus contamination (Lim et al., 2017).

Structure 25, 1–12.e1–e3, September 5, 2017 e1


METHOD DETAILS Temperature Perturbations of 2D22-DENV2 DENV2UN and DENV2EXP were produced by incubating purified DENV2 at 28 C and 37 C for 30 min respectively. The Fab fragment of 2D22 was expressed as a recombinant antibody fragment in 293F cell culture and purified by FPLC. All 2D22-DENV2 complexes were formed from incubation of purified DENV2 NGC (0.25 mg/mL) and Fab 2D22 (2.6 mg/mL) in a ratio of one E-protein: one 2D22 and subjected to the three temperature perturbation analyses described below: i) 2D22-DENV2UN at 28 C 2D22-DENV2UN was formed by incubating Fab 2D22 with DENV2UN in a ratio of one E-protein: one 2D22 at 28 C for 1 hr. ii) 2D22-DENV2EXP at 37 C 2D22-DENV2EXP was formed by incubating Fab 2D22 with DENV2EXP in a ratio of one E-protein: one 2D22 at 37 C for 1 hr. iii) 2D22-DENV2EXP-37 C 2D22-DENV2UN complex was first formed by incubating Fab 2D22 with DENV2UN in a ratio of one E-protein: one 2D22 at 28 C for 1 hr followed by inducing expansion of 2D22-DENV2UN with further incubation at 37 C for 30 min. HDXMS of 2D22-DENV2 Complexes and Fab 2D22 Deuterium exchange buffer was prepared by solubilizing lyophilized NTE (12 mM Tris-HCl, pH 8.0, 120 mM NaCl, 1 mM EDTA) buffer in 99.9% D2O. D2O NTE buffers were equilibrated separately at 28 and 37 C for 30 min prior to deuterium exchange reactions to ensure temperature consistency during HDX labelling reactions. Amide hydrogen/deuterium exchange of 2D22-DENV2 complexes were initiated by 10X dilution of 2D22-DENV2 complexes with temperature equilibrated D2O NTE buffers (final D2O concentration of 89.9%). HDX of 2D22-DENV2UN at 28 C was carried using D2O NTE buffers that were equilibrated at 28 C. HDX of 2D22-DENV2EXP, 2D22-DENV2UN-37 C and free 2D22 (2.6 mg/mL) were carried out using D2O NTE buffer equilibrated at 37 C. All deuterium exchange labelling was carried out for 1 min and performed in triplicates. Quenching HDX and Phospholipid Removal All deuterium exchange reactions were quenched by lowering the pHread to 2.5 upon addition of pre-chilled NaOH in GnHCl and Tris(2-carboxyethyl) phosphine-hydrochloride (TCEP-HCl) to achieve a final concentration of 1.5 M GnHCl and 0.25 M TCEP-HCl. Quenched reactions were maintained at 4 C on ice to minimize back exchange. Viral membrane phospholipids in the 2D22DENV2 complexes were removed by addition of 0.1 mg of titanium dioxide (TiO2) (Sigma Aldrich, St. Louis, MO) to the quenched deuterium exchange reaction mixture and incubated for 1 min with mixing every twice at intervals of 30 s(Hebling et al., 2010). TiO2 in the samples were removed with a 0.22 mm filter (Merck Millipore, Darmstadt, Germany) after 1 min of centrifugation at 13,000 rpm. Lipid removal using TiO2 resulted in an addition of 2 min to the sample processing time. Therefore, in the free Fab 2D22 samples without phospholipids, an additional two mins of incubation on ice was carried out to ensure identical post deuterium exchange reaction sample processing time with both the 2D22-DENV2 complexes and free Fab 2D22 samples. All deuterium exchange reactions were performed in triplicate, and the reported values for every peptide are an average of three independent reactions without correcting for deuterium back exchange. LC/MS of 2D22-DENV2 Complexes and Fab 2D22 Quenched samples of 2D22-DENV2 complexes and free 2D22 were injected into nano-UPLC HDX sample manager (Waters, Milford, MA), as previously described (Wales et al., 2008), and subjected to online pepsin digestion with a Waters Enzymate BEH pepsin (2.1 X 30 mm) column in 0.05% formic acid in water at 100 mL/min. Proteolyzed pepsin fragment peptides were trapped by a 2.1 X 5 mm C18 trap (ACQUITY BEH C18 VanGuard Pre-column, 1.7mm, Waters, Milford, MA) and eluted with an 8-40% gradient of acetonitrile in 0.1% formic acid at 40 mL/min into a reverse phase column (ACQUITY UPLC BEH C18 Column, 1.0 X 100 mm, 1.7 mm, Waters) by nanoACQUITY Binary Solvent Manager (Waters, Milford, MA). Peptides were ionized by electrospray into SYNAPT G2-Si mass spectrometer (Waters, Milford, MA) acquiring in MSE mode for detection and mass-measurements. 200 fmol/ml of [Glu1]-fibrinopeptide B ([Glu1]-Fib) was simultaneously injected into the mass spectrometer at a flow rate of 10 mL/min for continuous calibration during sample acquisition. QUANTIFICATION AND STATISTICAL ANALYSIS Pepsin Fragment Peptide Identification Peptides of C-, E- and M-protein from DENV2 NGC were identified by searching the mass spectra of the undeuterated samples of purified DENV2 NGC against DENV2 NGC structural protein database containing amino acid sequences of the C-, E- and M-proteins using PROTEIN LYNX GLOBAL SERVER version 3.0 (Waters, Milford, MA) software. Mass spectra of peptides from a single undeuterated DENV2 NGC samples with precursor ion mass tolerance of < 10 ppm, and products per amino acids of at least 0.2 with a minimum intensity of 5000 for both precursor and product ions were selected. Five undeuterated samples were collected and the final peptide list generated from peptides identified independently in at least 3 of the 5 undeuterated samples. Four glycopeptides (57-68, 57-69, 122-154,152-163) from DENV2 E-protein were identified using MSE (Xie et al., 2010), and the glycans were detected in several of the fragment peptides as indicated from our previous study (Lim et al., 2017). Therefore, these glycopeptides were e2 Structure 25, 1–12.e1–e3, September 5, 2017


included in the final peptide list. Peptides of C-, E- and M protein from 2D22-DENV2 complex were identified by searching the mass spectra of the undeuterated samples of the 2D22-DENV2 complexes against this peptide list. Mass spectra from three undeuterated samples of 2D22-DENV2 complex were collected and peptides were selected if they were identified independently in a minimum of 2 out of 3 undeuterated samples. Mass spectra of peptides from undeuterated free Fab 2D22 were searched against the database containing amino acid sequences of the heavy and light chain. Peptides were identified and selected only if they fulfill criteria described above. Three undeuterated samples of free Fab 2D22 were collected and the list of peptides used in this study were identified independently in a minimum of 2 out of 3 undeuterated samples. Peptides of Fab 2D22 heavy and light chain from 2D22-DENV2 complexes were identified by searching the mass spectra of the undeuterated samples of 2D22-DENV2 complexes against this peptide list. Mass spectra corresponding to peptides from three undeuterated samples of 2D22-DENV2 complex were selected if they were identified independently in a minimum of 2 out of 3 undeuterated samples. Deuterium Uptake Measurements in Peptides All deuterium exchange measurements were performed in triplicates and the deuterium uptake of each peptide were measured using DYNAMX Ver. 2.0 software (Waters, Milford, MA) by subtracting the average mass centroid of the peptide after 1 min of deuterium exchange with the average mass centroid of the corresponding undeuterated peptide. Determination of Deuterium Exchange Differences Differences in deuterium exchange for all peptides of the 2D22-DENV2 complexes under various temperature perturbations were determined by subtracting centroid masses of deuterated peptides between two experimental conditions and are represented in ‘‘difference plots’’. Deuterium exchange differences were measured for all identified peptides from DENV2 C-, E- and M-proteins and Fab 2D22 heavy and light chain and displayed from N to C-terminus in the respective difference plots. The standard deviations for deuterium uptake in all peptides were determined and a difference of 0.5 Da was used as the significance threshold for any differences in deuterium uptake across the two states compared and agrees with the observed standard errors measured in deuterated peptides (Houde et al., 2011).

Structure 25, 1–12.e1–e3, September 5, 2017 e3

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