Engineered human angiogenin mutations in the placental ribonuclease inhibitor complex for anticancer therapy: Insights from enhanced sampling simulations
Xiaojing Cong,1,2,3 Christian Cremer,4 Thomas Nachreiner,4 Stefan Barth,5,6* and Paolo Carloni1,2,3* 1
Computational Biophysics, German Research School for Simulation Sciences (Joint Venture of RWTH € lich), Ju €lich 52428, Germany Aachen University and Forschungszentrum Ju 2 €lich 52428, Germany, Computational Biomedicine Section, Institute for Advanced Simulations - 5 (IAS-5), Ju €lich Forschungszentrum Ju 3 €lich 52428, Germany, Computational Biomedicine Section, Institute for Neuroscience and Medicine - 9 (INM-9), Ju €lich Forschungszentrum Ju 4
Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Aachen 52074, Germany
5
Institute of Infectious Disease and Molecular Medicine (IDM), Faculty of Health Sciences, University of Cape Town, Cape Town 7925, South Africa
6
South African Research Chair in Cancer Biotechnology, Department of Integrative Biomedical Sciences, Faculty of Health Sciences, University of Cape Town, 7925 Cape Town, South Africa Received 14 March 2016; Accepted 20 April 2016 DOI: 10.1002/pro.2941 Published online 25 April 2016 proteinscience.org
Abstract: Targeted human cytolytic fusion proteins (hCFPs) represent a new generation of immunotoxins (ITs) for the specific targeting and elimination of malignant cell populations. Unlike conventional ITs, hCFPs comprise a human/humanized target cell-specific binding moiety (e.g., an antibody or a fragment thereof) fused to a human proapoptotic protein as the cytotoxic domain (effector domain). Therefore, hCFPs are humanized ITs expected to have low immunogenicity. This reduces side effects and allows long-term application. The human ribonuclease angiogenin (Ang) has been shown to be a promising effector domain candidate. However, the application of Angbased hCFPs is largely hampered by the intracellular placental ribonuclease inhibitor (RNH1). It rapidly binds and inactivates Ang. Mutations altering Ang’s affinity for RNH1 modulate the cytotoxicity of Ang-based hCFPs. Here we perform in total 2.7 ms replica-exchange molecular dynamics simulations to investigate some of these mutations—G85R/G86R (GGRRmut), Q117G (QGmut), and G85R/G86R/Q117G (GGRR/QGmut). GGRRmut turns out to perturb greatly the overall Ang-RNH1 interactions, whereas QGmut optimizes them. Combining QGmut with GGRRmut compensates the effects of the latter. Our results explain the in vitro finding that, while Ang GGRRmut-based hCFPs resist RNH1 inhibition remarkably, Ang WT- and Ang QGmut-based ones are similarly sensitive to RNH1 inhibition, whereas Ang GGRR/QGmut-based ones are only slightly resistant. This work may help design novel Ang mutants with reduced affinity for RNH1 and improved cytotoxicity. Additional Supporting Information may be found in the online version of this article. *Both authors contributed equally to this work. Grant sponsor: Deutsche Forschungsgemeinschaft (DFG, German Research Foundation); Grant number: CA 973/15-1. *Correspondence to: Paolo Carloni, Computational Biophysics, German Research School for Simulation Sciences (joint venture of €lich), Ju €lich 52428, Germany. E-mail: [email protected]; Stefan Barth: RWTH Aachen University and Forschungszentrum Ju E-mail: [email protected]
C 2016 The Protein Society Published by Wiley-Blackwell. V
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Keywords: targeted therapy; cytolytic fusion protein; apoptosis; angiogenin; ribonuclease inhibitor; mutagenesis; replica exchange; molecular dynamics
Introduction Immunotoxins (ITs) are recombinant fusion proteins consisting of a toxin genetically fused to a target cell-specific binding moiety. The latter is usually an antibody or a derivative thereof.1 The toxin moiety is typically derived from plants or bacteria, e.g., ricin A from Ricinus communis or Pseudomonas aeruginosa exotoxin A (ETA). The binding moieties are originated from mice, human or chimeric. Compared to the traditional cancer chemotherapy, ITs have the advantage that they are exceptionally specific for a distinctive target cell population. However, their non-human components are potentially immunogenic and can lead to several side effects upon repeated application.2 In addition, host development of neutralizing antibodies against these components promotes rapid clearance of the ITs from blood circulation and limits long-term treatment.3–5 To circumvent these problems, a new generation of ITs called targeted human cytolytic fusion proteins (hCFPs) is being developed. In these fusion proteins, humanized or fully human antibody fragments replace the murine counterparts and human proapoptotic proteins such as granzymes, ribonucleases (RNases) or microtubule-associated proteins (MAPs) substitute the bacterial/plant toxins to be the effector domain. Human CFPs are expected to be better tolerated than conventional ITs upon application.6 In this context, the human RNase angiogenin (Ang) is a promising candidate for the effector domain. Ang is a 14-kDa stress-activated enzyme with both angiogenic and ribonucleolytic activities. On the one hand, it enhances overall biogenesis, supports the synthesis of proteins necessary for blood vessel growth and promotes primary tumor development and metastasis.7 On the other hand, it induces translational repression by cleaving the 3’CAA terminus, the anticodon loop of tRNA as well as 5S, 18S and 28S rRNA.8–15 Using its ribonucleolytic activity, Ang has been successfully implemented as the effector domain in hCFPs.16–21 Unfortunately, therapeutic applications of Ang-based hCFPs are currently challenged by the ubiquitously expressed intracellular placental ribonuclease inhibitor RNH1.22 RNH1 has an extraordinary affinity for Ang in the femtomolar range23 and it is present in very high content (representing 0.01% of total intracellular proteins across almost all human cell types).22,24–28 RNH1 binding completely abolishes Ang’s activities.23,28–30 Hence, the efficacy of Angbased hCFPs would be greatly improved by evading RNH1, which undesirably mitigates most of the hCFPs delivered into the target cells.
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Some of us have shown that a dramatic decrease of Ang-RNH1 affinity by site-directed point mutations could significantly enhance the cytotoxicity of Ang-based hCFPs.31 We generated hCFPs composed of Ang variants genetically fused to the antiCD64 H22 single-chain variable fragment. The variants are: Ang G85R/G86R mutant (hereafter Ang GGRRmut), Ang Q117G mutant (Ang QGmut) and Ang G85R/G86R/Q117G mutant (Ang GGRR/QGmut). Ang GGRRmut is known to have 106-fold lower binding affinity for RNH1 than Ang wild type (WT),32 whereas Ang QGmut shows higher ribonucleolytic activity than Ang WT.33 To assess the inhibition of these hCFPs (denoted H22-Ang variants) by the ribonuclease inhibitor in vitro, we measured the biological activity of these hCFPs in HL-60 cells and human M1 macrophages (hM1U) (Supporting Information).34 In the in vitro experiments performed in the absence of ribonuclease inhibitor, we found more potent ribonuleolytic activity of both H22-Ang QGmut and H22-Ang GGRR/QGmut in comparison to H22Ang WT, whereas H22-Ang GGRRmut exhibited no significant difference. In the presence of ribonuclease inhibitor, however, H22-Ang GGRRmut showed the strongest ribonucleolytic activity, while the other hCFPs variants were significantly inhibited resulting in comparably lower efficiency (Supporting Information Fig. S1). Consistently, in both HL-60 cells and hM1U, H22-Ang GGRRmut showed remarkably lower EC50 values than H22-Ang WT (Supporting Information Fig. S2).34 However, since H22-Ang QGmut and H22-Ang GGRR/QGmut were sensitive to the ribonuclease inhibitor, they were only slightly more cytotoxic than H22-Ang WT (Supporting Information Fig. S2).31,34 Providing the molecular basis of the mutational effects on Ang’s affinity for RNH1 may help design new Ang variants with improved cytotoxicity. Here we predict the structure and the conformational flexibility of Ang WT, Ang GGRRmut, Ang QGmut and Ang GGRR/QGmut, each in complex with RNH1 in aqueous solution, by replica-exchange molecular dynamics (MD) simulations. The RNH1-Ang WT complex in aqueous solution turns out to preserve the X-ray structure in the solid state.35 RNH1-Ang GGRRmut experiences the largest fluctuations and a number of intermolecular interactions are disrupted. The results are consistent with the remarkable decrease in affinity induced by the GGRR mutation.32 RNH1-Ang QGmut shows the smallest fluctuations and forms the most extensive intermolecular interactions among the four variants. This is consistent with the in vitro finding that the Ang QGmut-based
Human Angiogenin Mutants Binding with RNH1
Figure 1. Representative structure of the RNH1-Ang WT trajectory (green) superimposed on the X-ray structure. The red to blue color indicates low to high Debye–Waller factors in the X-ray structure.35 The representative structure is obtained by cluster analysis of the trajectory using 2.5-A˚ cutoff on Ang Ca’s RMSD. It corresponds to the middle structure of the most populated cluster (including 54% of the frames).
hCFP is sensitive towards RNH1. We find that combining QGmut with GGRRmut enables Ang to recover the interactions with RNH1 that are otherwise disrupted by GGRRmut. Also these findings are fully consistent with in vitro data, which indicate that Ang GGRR/QGmut has much higher affinity for RNH1 than Ang GGRRmut.31,34
Results and Discussion RNH1-Ang WT in aqueous solution The RNH1-Ang WT complex is similar to the X-ray structure, as shown by a superimposition of the latter with the most populated conformer cluster in our simulations (Fig. 1). Ang binds in the center of the RNH1 ‘horseshoe’-shaped structure, forming extensive contacts with the C-terminal fraction of RNH1. Ang helix 2 and residues 85-59 interact with the Nterminal fraction of RNH1 and the middle of the horseshoe inner surface, respectively. The Ca’s ˚ and RMSD of RNH1 and Ang are 2.0 6 0.3 A
˚ , respectively (Supporting Information 3.0 6 0.6 A Fig. S3). Not unexpectedly, the C-terminus of RNH1 (Residues 1–5) is more flexible in solution than in the solid state, where it forms crystal-packing contacts.35 The Ca’s RMSF values (Fig. 2) correlate fairly well with the Debye–Waller factors of the Xray structure. In particular, loops and the N- and Ctermini of Ang feature relatively high RSMF and Debye–Waller values (Fig. 1). The RNH1 backbone is more rigid than that of Ang (Fig. 2). The intermolecular and intramolecular interactions formed by residues AngG85, AngG86, and Ang Q117 are analyzed in detail here because these residues are mutated in this study. In solution, Ang G85 and AngG86 interact largely with water molecules. This could be the case also for the X-ray structure in which likely not all the water molecules are resolved. AngG86 here also forms hydrogen bond (H-bond) with RNH1Q346 side chain and, to a lesser extent, with RNH1S289 side chain [Supporting Information Table S2, Fig. 3(A)]. In the X-ray structure
Figure 2. Ca’s RMSF values of (A) RNH1 and (B) the Ang variants, in RNH1-Ang WT (black), RNH1-Ang GGRRmut (blue), RNHAng QGmut (green), and RNH1-Ang GGRR/QGmut (red).
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Figure 3. Representative structures (from a cluster analysis) of (A) RNH1-Ang WT, (B) RNH1-Ang GGRRmut, (C) RNH1-Ang QGmut, and (D) RNH1-Ang GGRR/QGmut around Ang Residues 85–89. RNH1 and Ang backbones are shown in gray and yellow ribbons, respectively. Residues discussed in the main text are shown in sticks. Magenta dashed lines indicate H-bonds. Water molecules are not shown for clarity. Ang
G86 appears to interact only with RNH1S289 side chain. In solution, AngQ117 backbone is largely solvent-exposed. It also forms H-bonds with AngF120 and AngR121 backbones (Supporting Information Table S2). In the X-ray structure, it forms a waterbridged H-bond with RNH1D435 backbone and direct H-bonds with AngF120 and AngR121 backbones. Ang Q117 side chain forms H-bond with AngT44 backbone both in solution [Supporting Information Table S2, Fig. 4(A)] and in the X-ray structure. In solution, it forms H-bonds also with RNH1D435 side chain and Ang I42 backbone. In the X-ray structure, water molecules interacting with the two proteins are not all resolved, thus the interactions described here are putative.
RNH1-Ang GGRRmut in aqueous solution The overall RNH1 structure is similar to the X-ray ˚ , Supporting Inforstructure (Ca’s RMSD 2.1 6 0.5 A mation Fig. S3). Its conformational flexibility is similar to that in RNH1-Ang WT [Fig. 2(A)]. However, ˚, Ang GGRRmut shows larger Ca’s RMSD (3.4 6 0.8 A Supporting Information Fig. S3) and Ca’s RMSF (Fig. 2B) than Ang WT. Not unexpectedly, conformational rearrangements occur around the Ang mutation site (residues 85-89). Moreover, they also occur in distant regions from the mutation site, namely around Ang C-terminus (residues 117–123) and helix 2 (residues
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23–33). This indicates mutation-induced long-range effects on the overall complex structure (Fig. 5). Ang GGRRmut residues 85-89 increase their bend conformation by 33% relative to that in Ang WT (Table I). The backbone dihedrals of the mutated residues differ from those of Ang WT (Supporting Information Fig. S4). AngR85 and AngR86 backbone units are largely solvent exposed. In contrast to RNH1-Ang WT, AngG86 forms a H-bond with RNH1 Q346 side chain (Fig. 3). AngR85 side chain forms salt bridges with RNH1E149 and, to a lesser extent, with RNH1E206, while AngR86 side chain forms salt bridge with RNH1E206 and, to a lesser extent, with RNH1E264 [Fig. 3(B), Supporting Information Table S3]. The hydrophobic parts of R85 and R86 side chains form van der Waals contacts with the surrounding residues. While AngS87 and AngW89 maintain similar side-chain conformations to those in RNH1-Ang WT, AngP88 interacts more weakly with RNH1W375 than in RNH1-Ang WT (Supporting Information Table S4). The C-terminus of Ang GGRRmut shows higher 310-helix content than that in Ang WT and lower ahelix content (Table I). The C-terminus partially dissociates from RNH1 and the salt-bridge/H-bond network present in RNH1-Ang WT is largely disrupted (Fig. 4, Supporting Information Tables S2 and S3). The rearrangements appear to destabilize the
Human Angiogenin Mutants Binding with RNH1
Figure 4. Salt bridge and H-bond contacts (magenta dashed lines) around the C-terminus of Ang. The RNH1 backbone is shown in gray ribbon. The Ang backbone is shown in cartoon and colored by secondary structure. Positively and negatively charged residues that form salt bridges are labeled in blue and red, respectively. Main-chain and side-chain atoms are shown in sticks and balls, respectively. (A) RNH1-Ang WT preserves a salt-bridge/H-bond network throughout the simulation. (B) In RNH1-Ang GGRRmut, AngQ117 side chain swings between the original position (such as in RNH1-Ang WT) and a solventexposed one (shown here) where it forms an H-bond with RNH1N406. (C) RNH1-Ang QGmut maintains the same C-terminal conformation and intra-/intermolecular interactions as those in RNH1-Ang WT, except for the H-bonds involving the mutated Ang residue 117. (D) Ang GGRR/QGmut C-terminus adjusts its position with respect to RNH1 and forms stable salt bridges with the latter via AngR121 and AngR122.
Figure 5. Superimposition of representative structures of RNH1-Ang GGRRmut (blue) and RNH1-Ang WT (yellow) obtained by cluster analysis on the trajectories using 2.5 A˚ cutoff on Ang Ca’s RMSD. The most populated cluster of RNH1-Ang GGRRmut contains 39% of the frames. The mutation sites are indicated by red dots.
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Table I. Frequency of secondary structures (in %a) in selected regions that differ among the four Ang variants Region (residues) Helix 3 (50–58)
b-Strand 3 (69–72)
Residues 85–89
C-Terminus (117–121)
Ang Ang Ang Ang Ang Ang Ang Ang Ang Ang Ang Ang Ang Ang Ang Ang
Variant
a-Helix
WT GGRRmut QGmut GGRR/QGmut WT GGRRmut QGmut GGRR/QGmut WT GGRRmut QGmut GGRR/QGmut WT GGRRmut QGmut GGRR/QGmut
81 78 74 92
310-Helix
b-Sheet
(16) (17) (16) (14)
b-Turn
Bend
11 (11) 85 85 87 90
(15) (14) (13) (14)
15 (15) 13 (14) 13 (13) 61 81 67 30
53 37 76 30
(40) (40) (18) (40)
Coil
17 (30) 32 (38) 37 (38)
20 14 11 28
(20) (15) (15) (15)
35 19 32 70
(18) (15) (14) (15)
(24) (21) (13) (28)
Those with frequency
Xiaojing Cong,1,2,3 Christian Cremer,4 Thomas Nachreiner,4 Stefan Barth,5,6* and Paolo Carloni1,2,3* 1
Computational Biophysics, German Research School for Simulation Sciences (Joint Venture of RWTH € lich), Ju €lich 52428, Germany Aachen University and Forschungszentrum Ju 2 €lich 52428, Germany, Computational Biomedicine Section, Institute for Advanced Simulations - 5 (IAS-5), Ju €lich Forschungszentrum Ju 3 €lich 52428, Germany, Computational Biomedicine Section, Institute for Neuroscience and Medicine - 9 (INM-9), Ju €lich Forschungszentrum Ju 4
Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Aachen 52074, Germany
5
Institute of Infectious Disease and Molecular Medicine (IDM), Faculty of Health Sciences, University of Cape Town, Cape Town 7925, South Africa
6
South African Research Chair in Cancer Biotechnology, Department of Integrative Biomedical Sciences, Faculty of Health Sciences, University of Cape Town, 7925 Cape Town, South Africa Received 14 March 2016; Accepted 20 April 2016 DOI: 10.1002/pro.2941 Published online 25 April 2016 proteinscience.org
Abstract: Targeted human cytolytic fusion proteins (hCFPs) represent a new generation of immunotoxins (ITs) for the specific targeting and elimination of malignant cell populations. Unlike conventional ITs, hCFPs comprise a human/humanized target cell-specific binding moiety (e.g., an antibody or a fragment thereof) fused to a human proapoptotic protein as the cytotoxic domain (effector domain). Therefore, hCFPs are humanized ITs expected to have low immunogenicity. This reduces side effects and allows long-term application. The human ribonuclease angiogenin (Ang) has been shown to be a promising effector domain candidate. However, the application of Angbased hCFPs is largely hampered by the intracellular placental ribonuclease inhibitor (RNH1). It rapidly binds and inactivates Ang. Mutations altering Ang’s affinity for RNH1 modulate the cytotoxicity of Ang-based hCFPs. Here we perform in total 2.7 ms replica-exchange molecular dynamics simulations to investigate some of these mutations—G85R/G86R (GGRRmut), Q117G (QGmut), and G85R/G86R/Q117G (GGRR/QGmut). GGRRmut turns out to perturb greatly the overall Ang-RNH1 interactions, whereas QGmut optimizes them. Combining QGmut with GGRRmut compensates the effects of the latter. Our results explain the in vitro finding that, while Ang GGRRmut-based hCFPs resist RNH1 inhibition remarkably, Ang WT- and Ang QGmut-based ones are similarly sensitive to RNH1 inhibition, whereas Ang GGRR/QGmut-based ones are only slightly resistant. This work may help design novel Ang mutants with reduced affinity for RNH1 and improved cytotoxicity. Additional Supporting Information may be found in the online version of this article. *Both authors contributed equally to this work. Grant sponsor: Deutsche Forschungsgemeinschaft (DFG, German Research Foundation); Grant number: CA 973/15-1. *Correspondence to: Paolo Carloni, Computational Biophysics, German Research School for Simulation Sciences (joint venture of €lich), Ju €lich 52428, Germany. E-mail: [email protected]; Stefan Barth: RWTH Aachen University and Forschungszentrum Ju E-mail: [email protected]
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Keywords: targeted therapy; cytolytic fusion protein; apoptosis; angiogenin; ribonuclease inhibitor; mutagenesis; replica exchange; molecular dynamics
Introduction Immunotoxins (ITs) are recombinant fusion proteins consisting of a toxin genetically fused to a target cell-specific binding moiety. The latter is usually an antibody or a derivative thereof.1 The toxin moiety is typically derived from plants or bacteria, e.g., ricin A from Ricinus communis or Pseudomonas aeruginosa exotoxin A (ETA). The binding moieties are originated from mice, human or chimeric. Compared to the traditional cancer chemotherapy, ITs have the advantage that they are exceptionally specific for a distinctive target cell population. However, their non-human components are potentially immunogenic and can lead to several side effects upon repeated application.2 In addition, host development of neutralizing antibodies against these components promotes rapid clearance of the ITs from blood circulation and limits long-term treatment.3–5 To circumvent these problems, a new generation of ITs called targeted human cytolytic fusion proteins (hCFPs) is being developed. In these fusion proteins, humanized or fully human antibody fragments replace the murine counterparts and human proapoptotic proteins such as granzymes, ribonucleases (RNases) or microtubule-associated proteins (MAPs) substitute the bacterial/plant toxins to be the effector domain. Human CFPs are expected to be better tolerated than conventional ITs upon application.6 In this context, the human RNase angiogenin (Ang) is a promising candidate for the effector domain. Ang is a 14-kDa stress-activated enzyme with both angiogenic and ribonucleolytic activities. On the one hand, it enhances overall biogenesis, supports the synthesis of proteins necessary for blood vessel growth and promotes primary tumor development and metastasis.7 On the other hand, it induces translational repression by cleaving the 3’CAA terminus, the anticodon loop of tRNA as well as 5S, 18S and 28S rRNA.8–15 Using its ribonucleolytic activity, Ang has been successfully implemented as the effector domain in hCFPs.16–21 Unfortunately, therapeutic applications of Ang-based hCFPs are currently challenged by the ubiquitously expressed intracellular placental ribonuclease inhibitor RNH1.22 RNH1 has an extraordinary affinity for Ang in the femtomolar range23 and it is present in very high content (representing 0.01% of total intracellular proteins across almost all human cell types).22,24–28 RNH1 binding completely abolishes Ang’s activities.23,28–30 Hence, the efficacy of Angbased hCFPs would be greatly improved by evading RNH1, which undesirably mitigates most of the hCFPs delivered into the target cells.
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Some of us have shown that a dramatic decrease of Ang-RNH1 affinity by site-directed point mutations could significantly enhance the cytotoxicity of Ang-based hCFPs.31 We generated hCFPs composed of Ang variants genetically fused to the antiCD64 H22 single-chain variable fragment. The variants are: Ang G85R/G86R mutant (hereafter Ang GGRRmut), Ang Q117G mutant (Ang QGmut) and Ang G85R/G86R/Q117G mutant (Ang GGRR/QGmut). Ang GGRRmut is known to have 106-fold lower binding affinity for RNH1 than Ang wild type (WT),32 whereas Ang QGmut shows higher ribonucleolytic activity than Ang WT.33 To assess the inhibition of these hCFPs (denoted H22-Ang variants) by the ribonuclease inhibitor in vitro, we measured the biological activity of these hCFPs in HL-60 cells and human M1 macrophages (hM1U) (Supporting Information).34 In the in vitro experiments performed in the absence of ribonuclease inhibitor, we found more potent ribonuleolytic activity of both H22-Ang QGmut and H22-Ang GGRR/QGmut in comparison to H22Ang WT, whereas H22-Ang GGRRmut exhibited no significant difference. In the presence of ribonuclease inhibitor, however, H22-Ang GGRRmut showed the strongest ribonucleolytic activity, while the other hCFPs variants were significantly inhibited resulting in comparably lower efficiency (Supporting Information Fig. S1). Consistently, in both HL-60 cells and hM1U, H22-Ang GGRRmut showed remarkably lower EC50 values than H22-Ang WT (Supporting Information Fig. S2).34 However, since H22-Ang QGmut and H22-Ang GGRR/QGmut were sensitive to the ribonuclease inhibitor, they were only slightly more cytotoxic than H22-Ang WT (Supporting Information Fig. S2).31,34 Providing the molecular basis of the mutational effects on Ang’s affinity for RNH1 may help design new Ang variants with improved cytotoxicity. Here we predict the structure and the conformational flexibility of Ang WT, Ang GGRRmut, Ang QGmut and Ang GGRR/QGmut, each in complex with RNH1 in aqueous solution, by replica-exchange molecular dynamics (MD) simulations. The RNH1-Ang WT complex in aqueous solution turns out to preserve the X-ray structure in the solid state.35 RNH1-Ang GGRRmut experiences the largest fluctuations and a number of intermolecular interactions are disrupted. The results are consistent with the remarkable decrease in affinity induced by the GGRR mutation.32 RNH1-Ang QGmut shows the smallest fluctuations and forms the most extensive intermolecular interactions among the four variants. This is consistent with the in vitro finding that the Ang QGmut-based
Human Angiogenin Mutants Binding with RNH1
Figure 1. Representative structure of the RNH1-Ang WT trajectory (green) superimposed on the X-ray structure. The red to blue color indicates low to high Debye–Waller factors in the X-ray structure.35 The representative structure is obtained by cluster analysis of the trajectory using 2.5-A˚ cutoff on Ang Ca’s RMSD. It corresponds to the middle structure of the most populated cluster (including 54% of the frames).
hCFP is sensitive towards RNH1. We find that combining QGmut with GGRRmut enables Ang to recover the interactions with RNH1 that are otherwise disrupted by GGRRmut. Also these findings are fully consistent with in vitro data, which indicate that Ang GGRR/QGmut has much higher affinity for RNH1 than Ang GGRRmut.31,34
Results and Discussion RNH1-Ang WT in aqueous solution The RNH1-Ang WT complex is similar to the X-ray structure, as shown by a superimposition of the latter with the most populated conformer cluster in our simulations (Fig. 1). Ang binds in the center of the RNH1 ‘horseshoe’-shaped structure, forming extensive contacts with the C-terminal fraction of RNH1. Ang helix 2 and residues 85-59 interact with the Nterminal fraction of RNH1 and the middle of the horseshoe inner surface, respectively. The Ca’s ˚ and RMSD of RNH1 and Ang are 2.0 6 0.3 A
˚ , respectively (Supporting Information 3.0 6 0.6 A Fig. S3). Not unexpectedly, the C-terminus of RNH1 (Residues 1–5) is more flexible in solution than in the solid state, where it forms crystal-packing contacts.35 The Ca’s RMSF values (Fig. 2) correlate fairly well with the Debye–Waller factors of the Xray structure. In particular, loops and the N- and Ctermini of Ang feature relatively high RSMF and Debye–Waller values (Fig. 1). The RNH1 backbone is more rigid than that of Ang (Fig. 2). The intermolecular and intramolecular interactions formed by residues AngG85, AngG86, and Ang Q117 are analyzed in detail here because these residues are mutated in this study. In solution, Ang G85 and AngG86 interact largely with water molecules. This could be the case also for the X-ray structure in which likely not all the water molecules are resolved. AngG86 here also forms hydrogen bond (H-bond) with RNH1Q346 side chain and, to a lesser extent, with RNH1S289 side chain [Supporting Information Table S2, Fig. 3(A)]. In the X-ray structure
Figure 2. Ca’s RMSF values of (A) RNH1 and (B) the Ang variants, in RNH1-Ang WT (black), RNH1-Ang GGRRmut (blue), RNHAng QGmut (green), and RNH1-Ang GGRR/QGmut (red).
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Figure 3. Representative structures (from a cluster analysis) of (A) RNH1-Ang WT, (B) RNH1-Ang GGRRmut, (C) RNH1-Ang QGmut, and (D) RNH1-Ang GGRR/QGmut around Ang Residues 85–89. RNH1 and Ang backbones are shown in gray and yellow ribbons, respectively. Residues discussed in the main text are shown in sticks. Magenta dashed lines indicate H-bonds. Water molecules are not shown for clarity. Ang
G86 appears to interact only with RNH1S289 side chain. In solution, AngQ117 backbone is largely solvent-exposed. It also forms H-bonds with AngF120 and AngR121 backbones (Supporting Information Table S2). In the X-ray structure, it forms a waterbridged H-bond with RNH1D435 backbone and direct H-bonds with AngF120 and AngR121 backbones. Ang Q117 side chain forms H-bond with AngT44 backbone both in solution [Supporting Information Table S2, Fig. 4(A)] and in the X-ray structure. In solution, it forms H-bonds also with RNH1D435 side chain and Ang I42 backbone. In the X-ray structure, water molecules interacting with the two proteins are not all resolved, thus the interactions described here are putative.
RNH1-Ang GGRRmut in aqueous solution The overall RNH1 structure is similar to the X-ray ˚ , Supporting Inforstructure (Ca’s RMSD 2.1 6 0.5 A mation Fig. S3). Its conformational flexibility is similar to that in RNH1-Ang WT [Fig. 2(A)]. However, ˚, Ang GGRRmut shows larger Ca’s RMSD (3.4 6 0.8 A Supporting Information Fig. S3) and Ca’s RMSF (Fig. 2B) than Ang WT. Not unexpectedly, conformational rearrangements occur around the Ang mutation site (residues 85-89). Moreover, they also occur in distant regions from the mutation site, namely around Ang C-terminus (residues 117–123) and helix 2 (residues
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23–33). This indicates mutation-induced long-range effects on the overall complex structure (Fig. 5). Ang GGRRmut residues 85-89 increase their bend conformation by 33% relative to that in Ang WT (Table I). The backbone dihedrals of the mutated residues differ from those of Ang WT (Supporting Information Fig. S4). AngR85 and AngR86 backbone units are largely solvent exposed. In contrast to RNH1-Ang WT, AngG86 forms a H-bond with RNH1 Q346 side chain (Fig. 3). AngR85 side chain forms salt bridges with RNH1E149 and, to a lesser extent, with RNH1E206, while AngR86 side chain forms salt bridge with RNH1E206 and, to a lesser extent, with RNH1E264 [Fig. 3(B), Supporting Information Table S3]. The hydrophobic parts of R85 and R86 side chains form van der Waals contacts with the surrounding residues. While AngS87 and AngW89 maintain similar side-chain conformations to those in RNH1-Ang WT, AngP88 interacts more weakly with RNH1W375 than in RNH1-Ang WT (Supporting Information Table S4). The C-terminus of Ang GGRRmut shows higher 310-helix content than that in Ang WT and lower ahelix content (Table I). The C-terminus partially dissociates from RNH1 and the salt-bridge/H-bond network present in RNH1-Ang WT is largely disrupted (Fig. 4, Supporting Information Tables S2 and S3). The rearrangements appear to destabilize the
Human Angiogenin Mutants Binding with RNH1
Figure 4. Salt bridge and H-bond contacts (magenta dashed lines) around the C-terminus of Ang. The RNH1 backbone is shown in gray ribbon. The Ang backbone is shown in cartoon and colored by secondary structure. Positively and negatively charged residues that form salt bridges are labeled in blue and red, respectively. Main-chain and side-chain atoms are shown in sticks and balls, respectively. (A) RNH1-Ang WT preserves a salt-bridge/H-bond network throughout the simulation. (B) In RNH1-Ang GGRRmut, AngQ117 side chain swings between the original position (such as in RNH1-Ang WT) and a solventexposed one (shown here) where it forms an H-bond with RNH1N406. (C) RNH1-Ang QGmut maintains the same C-terminal conformation and intra-/intermolecular interactions as those in RNH1-Ang WT, except for the H-bonds involving the mutated Ang residue 117. (D) Ang GGRR/QGmut C-terminus adjusts its position with respect to RNH1 and forms stable salt bridges with the latter via AngR121 and AngR122.
Figure 5. Superimposition of representative structures of RNH1-Ang GGRRmut (blue) and RNH1-Ang WT (yellow) obtained by cluster analysis on the trajectories using 2.5 A˚ cutoff on Ang Ca’s RMSD. The most populated cluster of RNH1-Ang GGRRmut contains 39% of the frames. The mutation sites are indicated by red dots.
Cong et al.
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Table I. Frequency of secondary structures (in %a) in selected regions that differ among the four Ang variants Region (residues) Helix 3 (50–58)
b-Strand 3 (69–72)
Residues 85–89
C-Terminus (117–121)
Ang Ang Ang Ang Ang Ang Ang Ang Ang Ang Ang Ang Ang Ang Ang Ang
Variant
a-Helix
WT GGRRmut QGmut GGRR/QGmut WT GGRRmut QGmut GGRR/QGmut WT GGRRmut QGmut GGRR/QGmut WT GGRRmut QGmut GGRR/QGmut
81 78 74 92
310-Helix
b-Sheet
(16) (17) (16) (14)
b-Turn
Bend
11 (11) 85 85 87 90
(15) (14) (13) (14)
15 (15) 13 (14) 13 (13) 61 81 67 30
53 37 76 30
(40) (40) (18) (40)
Coil
17 (30) 32 (38) 37 (38)
20 14 11 28
(20) (15) (15) (15)
35 19 32 70
(18) (15) (14) (15)
(24) (21) (13) (28)
Those with frequency
