Structure and function of retroviral proteases.

ANNUAL REVIEWS

Biophys. Biophys. Chern. 1991.20:299-320 © 1991 by Annual Reviews Inc. All rights reserved

Annu. Rev.

Cupyright

Further

Quick links to online content

STRUCTURE AND FUNCTION Annu. Rev. Biophys. Biophys. Chem. 1991.20:299-320. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 05/28/15. For personal use only.

OF RETROVIRAL PROTEASES P. M. D. Fitzgerald and .T. P. Springer

Department of Biophysical Chemistry, Merck Sharp and Dohme Research Laboratories, Rahway, New Jersey 07065 KEY WORDS:

aspartyl protease, X-ray diffraction, symmetry, AIDS

CONTENTS

. .............................

299

...... ...................................... .. ... .. ... . . ... .. ... . ......... . ... Isolation ...................................... ................. ....... ... . ............................. .... ................ Synthesis .................................................................................................................. Expression ... ... ... .................... .................................. ..................................

302 302 303 303

SUBSTRATE SPECIFICITy..................................................................................................

304

INHIBITION ....................................................................................................................

306

THREE-DIMENSIONAL STRUCTURES OF NATIVE PROTEASES.................................................

308

RS V Protease...................................... ....... .............................................................. HI V-l Protease ................................................ ..... ... ... ... .......................................... Comparisons with Structures of Native Monomeric Aspartyl Proteases .. . Implicationsfor Structures in Other Retroviral Classes.............................................

308

311

... ..... ........... . ......... . .. .. . .. ...... HI V-l Protease ............................... ........................................... . . ......... . . . . . . . . . . . . . . . . . . . Comparison with Structures of Inhibited Monomeric Aspartyl Proteases ..................

312 314

MUTAGENESIS ...... .. .. . .

. . . . .. .... ... ... .. ....... ...... . ............... ....... ... ...........................................

315

ROLE OF SYMMETRY IN RETROVIRAL PROTEASE STRUCTURES ........... ................... .............

316 316

PERSPECTIVES AND OVERVIEW ....................................................... ...

301

PRIMARY STRUCTURE ................................. . ISOLATION AND CHARACTERIZATION

.

.........

.

.

......... . .......

THREE-DIMENSIONAL STRUCTURES OF INHIBITED COMPLEXES

. Genetics................ ............. ...... ................................................................................. Deviations from Ahsolute Symmetry ..... ...... .................................... ..

.

.

.

. .

. ......... ... ....... .. .. .....

SUMMARY AND FUTURE DIRECTIONS ......... ..... ................... ... ........ ..

310 311

312

317 317

PERSPECTIVES AND OVERVIEW

Considerable progress has been made in understanding the virology, molecular biology, and biochemistry of the class of viruses known as 299 0883-9182/91/0610-0299$02.00

Annu. Rev. Biophys. Biophys. Chem. 1991.20:299-320. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 05/28/15. For personal use only.

300

FITZGERALD & SPRINGER

retroviridae. The discovery that human immunodeficiency virus, the causa tive agent of acquired immunodeficiency syndrome (AIDS), is a retrovirus has intensified efforts to understand key steps in the life cycle of retro viruses. The goal of these efforts is to interrupt those processes, and thus the spread of infection, through therapeutic intervention. This review focuses on the role that the retroviral-encoded proteases play in viral propagation, on studies to understand the structure and function of these enzymes, and on the search for cffective inhibitors to be used in the treatment of retroviral-associated diseases. The biochemistry of viral pro teases has recently been reviewed (37), and interest in viral proteases as targets in chemotherapy has already led to a meeting on that topic (44). Analysis of viral DNA has enabled identification of proteases from many types of retroviruses, and a number of these have been isolated, purified, and characterized. However, by far the most well-studied proteases have been from Rous sarcoma virus (RSV) and the closely related avian myelo blastosis virus (AMV) and from human immunodeficiency virus types 1 and 2 (HIV I and HIV-2). This review focuses on these enzymes. The genomic material of retroviruses is single-stranded RNA. After viral infection of susceptible cells, the RNA is transcribed first to a RNA�DNA heteroduplex and subsequently to a DNA homoduplex that is enzymatically inserted into the host cell genome. During viral propagation, this proviral DNA is transcribed to RNA, which can serve either as genomic RNA for progeny virions or as messenger RNA for the translation of viral proteins. Viral proteins are translated as polyproteins, and pro cessing of these polyproteins into mature viral structural proteins and enzymes requires the activity of a viral-encoded protease. Figure 1 illus trates the organization of open reading frames in the genomes of four characteristic classes of retroviruses. As can be seen in that figure, the viral protease can be coded for in the gag or pol open reading frame, or in a reading frame between the two; various mechanisms effect the occasional translation of the gag-pol or gag-prt-pol fusion polyproteins. The single frameshifting mechanism of the HIV-I system has been studied in some detail (28). The frameshift is believed to involve a RNA sequence of UUUA; the ribosomes shift down one frame from a leucine (UUA) to a phenylalanine (UUU) codon. Jacks et al (28) have proposed that a stem loop structure 3' to the frameshift site may increase the frequency of the frameshifting event. Earlier studies in the RSV system implicate a similar mechanism (30), and the double frameshifting retroviruses [e.g. mouse mammary tumor virus (29) and human T-cell leukemia virus type I (HTLV-I) (69)] appear to utilize this mechanism downstream of the prt open reading frame. A second mechanism, involving a polyadenine nucleo tide sequence, has been described for the frameshifting event that occurs -

RETROVIRAL PROTEASES

301

RSV gag

pol

I��r i

MMTV

gag

prt

I l'rll


i

pol

i

MoMuLV

gag

pol

If�_�'

i

HIV-1 gag

I

i

I

fi1¥#M

pol

Figure 1

Organization of the protease domain with respect to the gag and pol open reading frames in the genome of several representative retroviruses. The protease coding region has been shaded. The diagrams are based on the nucleotide sequences for Rous sarcoma virus (RSV) (87), mouse mammary tumor virus (MMTV) (66), Moloney murine leukemia virus (MoMuLV) (90), and human immunodeficiency virus type I (HIV-I ) (68, 77, 83 , 1 04). Arrows indicate the approximate position of frameshifting events for RSV (30), MMTV (29), and HIV- I (28, 1 09). The approximate position of the termination codon readthrough for MoM uLV is indicated by an arrow ( 1 1 2) .

upstream of the prt open reading frame in these viruses (29, 69)_ In the class of retroviruses that includes Moloney murine leukemia virus (MoM uLV) and feline leukemia virus, the gag and pol open reading frames are in register and separated by a single amber stop codon; the translation of the gag-pol polyprotein involves a suppression readthrough of this stop codon (I 11, 112)_ PRIMARY STRUCTURE

Proteases can be assigned to one of four categories, each of which contains characteristic primary sequence features, based on the nature of the cata lytic mechanism: serine proteases, sulfhydryl proteases, metalloproteases, and aspartyl proteases. The aspartyl-protease family of enzymes, which includes such well-studied enzymes as pepsin, renin, chymosin, cathepsin D, and cathepsin E, is characterized by a conserved Asp-Thr-Gly sequence that occurs twice in each enzyme sequence of 320-335 amino acids_ Analy sis of the primary structure of pepsin (89) indicated that the sequences of

302

FITZGERALD

& SPRINGER


the amino- and carboxyl-terminal halves of the enzyme can be aligned with one another, and suggested that pepsin may have arisen by gene duplication from a primordial dimeric ancestor. Structural studies of three

aspartyl proteases of fungal origin, endothiapepsin (4), penicillopepsin (31), and rhizopuspepsin (95), confirmed the organization of these enzymes into two similarly folded domains with the active site at the interface between the two domains. Comparisons of the structures of these enzymes revealed close structural homology (94, 97). Recent structural studies of two aspartyl proteases of mammalian origin, bovine chymosin (22) and human renin (91), have supported the structural similarity of this family of enzymes. The structure and function of aspartyl proteases has been

recently reviewed (11). When sequence information for retroviral proteases became available, researchers (98) noted that these proteases contained the Asp-Thr-Gly sequence known to be common to aspartyl proteases (with the occasional replacement of Ser for Thr). However, retroviral proteases are much smaller proteins than the pepsin-like aspartyl proteases, ranging in size from 99 amino acids for HIV-1 protease to approximately 125 amino acids for the largest. Thus, Pearl & Taylor (73) suggested that the active form of retroviral proteases would be a dimer and that this dimer might represent a remote point in the evolution of aspartyl proteases. ISOLATION AND CHARACTERIZATION Isolation

Most of the early investigations of the properties of retroviral proteases focused on the RSV-AMV system. In this system, unlike other retroviral systems, the protease resides at the 3' end of the gag gene product (Figure 1) and thus can be isolated in quantity from viral cultures. Purified prep arations of the pIS protein (so-called because of its molecular mass of 15,000 daltons) have proteolytic activity against the pr76 polyprotein precursor, producing products of the same size as the mature proteins isolated from intact viruses (14, 102, 103). The amino acid sequence of the AMV protease (pIS) was reported in 1981 (84); subsequently the proteases from bovine leukemia virus (BL V) (113), feline leukemia virus (111), and MoMuLV (112) were isolated and characterized by amino- and (in the case of BLV and MoMuLV) carboxyl terminal sequence analysis. The development of modern techniques of genetic isolation and cloning led to a burst of information about the DNA sequences, and hence the inferred protein sequences, of putative proteases from many types of retroviruses. In most cases, these proteins have never been isolated, but


303

are assumed to be proteases because of sequence homology with previously characterized retroviral proteases; as a result the proper amino and car boxyl termini of these proteins are uncertain.


Synthesis

Isolation of HIV-I protease from virus particles yields only small quantities of protease (51) and carries the risks associated with handling a deadly virus. One successful approach for obtaining sufficient quantities of H IV1 protease for enzymatic and structural characterization is total chemical synthesis. Nutt et al (71) showed that synthetic protease (NY5 strain) (1) could be folded into an active enzyme that behaved as a dimer on size exclusion chromatography. Schneider & Kent (86) synthesized a protease corresponding to the sequence of the SF 2 strain (83) of HIV-I , and Cope land & Oroszlan (7) synthesized the proteases from both HIV-I and HIV2. The synthetic proteases were shown either to cleave the natural substrate gag p55 into mature gag proteins (71, 86) or to cleave oligopeptides corresponding to the protease processing sites in the HIV fusion poly proteins (7, 10, 86). Expression

Constructs of AMV protease and upstream portions of the gag gene have been cloned and expressed; the protease correctly liberates itself from the precursor (42). The 372-base pair coding sequence for the protease can also he directly expressed (42). HIV-l protease has also been generated by cloning and expression. In several laboratories, portions of the pol polyprotein containing the pro tease-coding sequence as well as flanking amino and carboxyl sequences were cloned and expressed (3, 12, 23, 24, 46); the protease was found to process itself out of the expressed proteins, resulting in the accumulation of a mature I I ,OOO-dalton form of the protease as observed on SDS PAGE. Other constructs containing non-HIV genetic material linked to portions of the pol polyprotein also autoprocessed to produce an 11,000dalton protease (21, 67, 75). Subsequently, constructs that contained that coding region of HIV-l protease alone (9, 23) were also cloned and expressed. Genes for the 297 base pairs that code for HIV-I protease have been synthesized and then expressed (27, 55); these synthetic genes should allow for convenient mutagenesis of the protease. The proteases from several of these expression systems have been purified to apparent homo geneity; purification schemes that utilize HIV-1 protease inhibitors linked to a support to generate an affinity column have proved particularly convenient (16, 25).

304


SUBSTRATE SPECIFICITY

The sequences of naturally occurring protease cleavage sites have been studied to detect patterns of amino acid preferences. [We use the notation of Schechter & Berger (85), in which residues of the substrate are numbered Pj, P2, , Pn from the scissile bond towards the amino terminus and P;, Pl, . . . , P� from the scissile bond towards the carboxyl terminus. We also use the abbreviations for retroviral proteins proposed by Leis et al (49): membrane associated protein, MA; capsid protein, CA; nucleocapsid pro tein, NC; protease, PR; reverse transcriptase, RT; integrase, IN. ] An analysis of MA-CA (see Table 1) junctions in 17 retroviruses (72) showed that while many amino acids were tolerated at positions P4, P3, P2, and p), only four amino acids occurred at position PI (Phe, Tyr, Met, Leu), three at P; (Val, lIe, Leu), and one at P; (Pro). An analysis of cleavage sites in HIV-l, HIV-2, and simian immunodeficiency virus (26) led to the proposal that the cleavage sites in these viruses could be assigned to one of three classes. Sequences in class I have Phe at position PI and Pro at position P;; class 2 sequences have Arg at position P4 and Phe-Leu at positions P; P;; and class 3 sequences have Gin or Glu at position Pl' Preliminary substrate specificity studies of AMV protease using syn thetic peptides have led to the conclusion that high salt levels increase the rate of catalysis, that aromatic or bulky aliphatic residues are preferred in position PI> and that the central four residues of a substrate need to be considered together when evaluating amino acid preferences (40, 41,


• • •

93).

The specificity of HIV proteases towards their natural substrates has been probed using mutagenesis in recombinant systems. In one experiment (47), HIV- l protease was cloned into the pol polyprotein of HIV-2 and Table 1

Cleavage sites for HIV-1 protease in the gag and gag-pol polyproteins Sequenceb

Cleavage site" MA-CA CA-CA+ CA+-NC p9-term p6-PR PR-RT RT-IN

Gin His Asn Gly Thr Gly Gly

Val Lys Thr Arg Val Cys Ile

Ser Ala Ala Pro Ser Thr Arg

GIn Arg Thr Gly Phe Leu Lys

Asn Val Ile Asn Asn Asn Ile

Tyr Leu Met Phe Phe Phe Leu

* *

* * * * *

Pro Ala Met Leu Pro Pro Phe

Ile Glu Gin GIn Gin lie Leu

Val Ala Arg Ser lie Ser Asp

GIn Met Gly Arg Thr Pro Gly

Asn Ser Asn Pro Leu Ile Ile

Ile GIn Phe Glu Trp Glu Asp

------._----- -

"The nomenclature proposed by Leis et al (49) designates each of the gag and gag-pol proteins. Thc cleavage sites are as described by Kriiusslich et al (43) and Darke et al (10). h The asterisk indicates the position of the scissile bond.



305

vice versa. Processing of the polyproteins proceeded more slowly in these heterologous systems than in the corresponding homologous systems, indicating a preference of each protease for its natural cleavage targets. In another experiment (52), the residues in the p\ and P; positions of the p6PR, PR-RT, and RT-IN cleavage sites of the pol polyprotein were ran domly altered by mutagenesis. The RT-IN site was found to be completely intolerant of change; the PR-RT site tolerated a few changes; and the p6PR site (natural sequence Phe-Pro) was most forgiving-sequences as different as Gly-His and Ala-lie resulted in processing equivalent to that of the wild-type sequence. Kinetic studies of the catalysis of peptides corresponding to the natural substrates of HIV-I have shown that the p6-PR junction and the CA + NC junction are cleaved more readily than the other natural sites (10,43). Interestingly, these studies showed that the MA-CA junction, widely used as a template for substrate and inhibitor design (see below), was not one of the best substrates. Recently, several groups have reported studies in which specific sites on a template oligopeptide HIV-l substrate were varied to determine the preferences for amino acids at a given position. Konvalinka et al (39) varied amino acids at positions P3, P2, and p\ in the chromogenic substrate Ala-Thr-His-Gln-Val-Tyr*Nph-Val-Arg-Lys-Ala (Nph, 4-N02-phenyl alanine; asterisk, position of the scissile bond). They found that position P3 tolerated a variety of residues, but not proline, that position P2 showed a marked preference for p-branched amino acids (Val and lie), and that peptides containing Met, Phe, and Tyr at position PI were good substrates while peptides containing Arg or Glu at that position were not hydrolyzed. Variation of the residue at position PI in the substrate Lys-Ala-Arg-Val X*Nph-Glu-Ala-Met/Nle-NH2 (Nle, norleucine; X, position of residue varied) showed that a peptide containing Nle was the best substrate of those tested, followed closely by peptides containing Leu, Tyr, and Met at that position, while peptides containing lie and Val were poor substrates (79). Variation of the residue in the P2 position of the peptide Ser-Gln Asn-Tyr*Pro-X-Val showed that lie was preferred, followed by Leu and Ala; peptides with Phe and Gly were poor substrates, and peptides con taining Trp at that position were not cleaved (56). Tomaselli et al (99) showed that HIV-l protease cleaves between domains of a truncated Pseudomonas exotoxin molecule, but were sur prised to find that cleavage does not occur at the expected Leu-Glu Arg-Asn-Tyr*Pro-Thr-Gly sequence but at an adjacent Ser-Gly-Asp-Ala Leu*Leu-Glu-Arg-Asn sequence. They tested whether this resulted from higher-order conformational effects by studying the hydrolysis of isolated peptides with the same sequences. A peptide corresponding to the Leu*Leu

306


sequence was cleaved while one corresponding to the Tyr*Pro sequence was not, indicating that sequence, not conformation, determined cleavage.


INHIBITION

Inhibition by pepstatin A (isovaleryl-Val-Val-statine-Ala-statine) [statine, (4S,3S)-4-amino-3-hydroxy-6-methylheptanoic acid (101)] is a defining characteristic of the aspartyl-protease family of enzymes. Thus, when analysis of retroviral protease sequences implied that these enzymes were members of that family, the enzymes were tested for inhibition by pepstatin A. BLV, MoMuLV, HTLV-I proteases (35), and AMV protease (40) were all inhibited by pepstatin A, although at higher concentrations than those required to effectively inhibit the monomeric aspartyl proteases. Synthetic HIV-I protease (71, 86) and partially purified preparations of recombinant HIV-l protease (21, 24, 45, 80, 88) also showed pepstatin A inhibi tion. Inhibition constants in the range 0.7-1.4 11M were reported for purified recombinant and synthetic HIV-l protease (9, 15, 43, 71). Richards et al (80) found that acetyl-pepstatin (acetyl-Val-Val-statine Ala-statine) is a more potent inhibitor (Ki 20 nM) of HIV-1 protease than pepstatin A, and an even more potent (Ki 5 nM) inhibitor of HIV-2 protease (78), although the inhibition constant is highly depen dent on pH (optimum at pH 4.7) and ionic strength (optimum at 1 M NaCI). Because of the hope that effective and specific inhibition of HIV-I protease will provide a novel therapeutic approach to the treatment of AIDS, considerable effort has recently been focused on the design and synthesis of HIV-I protease inhibitors. One approach has been to modify natural substrates of the enzyme (Table 1) with one of the transition-state mimics illustrated in Figure 2. Dreyer et al (15) and Moore et al (65) modified hexa- or heptapeptides with sequences based on the MA-CA cleavage site (see Table 1) with each of the transition state mimics, obtain ing competitive inhibitors with Ki values in the range 18 nM to 40 pM. They found that the hydroxycthylcne isosteres (with the S absolute con figuration at the inhibitory hydroxyl group) were the most potent inhibi tors. Compounds in this series were later shown to inhibit the activity of HIV-1 protease in infected T lymphocytes and to block the spread of viral infection at concentrations of 25 to 100 11M (60). Tomasselli et al (100) studied hydroxyethylene-containing octapeptides based on the MA-CA cleavage site. In the most potent of these compounds, the scissile Tyr*Pro dipeptide, was replaced with LeUljJ[CH(OH)CH2]Val; this compound showed a Kj of < 10 nM for HIV-1 protease. A more recent report from the same group (58) described the compound Tba=

=



o

)-

307

0

N J-- � _\_./\ � OH

0

Figure 2 Examples of the substructure of a substrate for an aspartyl protease and of the substructures oftive classes of inhibitor designed to be mimics of the transition state. (Upper left) A hypotheti al substrate, Gly-Leu-Leu-Gly. (Upper right) A hypotheti l reduced

c

ca

A hypothetical statine-con taining inhibitor, Gly-Sta-Gly. Note that statine mimics a dipeptide. (Middle right) A hypothetical hydroxycthylcnc-containing inhibitor, Gly-Leuift[CH(OH)CHzlLeu-Gly. (Lower left) A hypothetical hydroxyethylamine-containing inhibitor, Gly-Leuift[CH (OH)CH N H 1Leu-Gly. (Lower right) A hypothetical phosphinic acid-- 10 ,uM for five human aspartyl proteases. In addition to the active site-directed inhibitors discussed above, inhibition of dimer formation might be an alternative method of con trolling protease activity (110). This approach has the advantage that a potent inhibitor would not have cross activity against the monomeric aspartyl proteases. THREE-DIMENSIONAL STRUCTURES OF NATIVE PROTEASES RSV Protease

The proposal that the retroviral proteases were dimeric (73) and each monomer contributed a single Asp-Thr(Ser)-Gly sequence to the active site was confirmed when RSV protease was crystallized (63) and its three dimensional structure determined (33, 62). Table 2 contains details of all of the crystallographic structure determinations discussed in this review. (In those structures with two monomers in the crystallographic asymmetric unit, we denote residues in one monomer as 1, 2, . . . , n and residues in the second monomer as 201, 202, ... , n. This convention is not necessarily that used by the original authors.) The two crystallographically independent monomers of RSV protease interact to form a nearly symmetric dimer as shown in Figure 3. A 1780 rotation superimposes the (J(-carbon atoms of one monomer on the equivalent atoms in the second monomer with a root mean square (rms) deviation of 0.4 A. Each monomer is composed of several /i-strands, one well-defined (J(-helix, and one helical turn. Three regions of each monomer participate in interactions that stabilize the dimer: the immediate active-site residues; the amino- and carboxyl-ter minal regions, which form a four-stranded antiparallel /i-sheet; and the side chains of residues Asp-41, Arg-l l l, and Arg-21O. A water molecule in the active-site region interacts with the two catalytic aspartic acid resi dues (Asp-37 and Asp-237). The only parts of the structure that are not well localized are the regions known as the flap. In the monomeric aspartyl proteases, the equivalent residues are flexible, adjusting their position to facilitate interaction with inhibitors (32). In the RSV protease crystal structure, which does not contain an inhibitor, the flaps probably have no fixed conformation. The structure of the closely related AMV protease has been determined in several laboratories, but to date only preliminary


Table 2

Structure determinations of retroviral proteases

Inhibitor

Enzyme

Enzyme

Space

Nominal

Cell dimensions

Number of

R

Bond

source

sequence

group

resolution

a

b

reflections

value

distances

2.0

88.95

88.95

17,835

0.144

0.022

References

RSV/AMV protease none

viral

RSV

P3121

78.90

33

HIV-I protease native enzyme none

recombinant

XHB2

P41212

2.7

2,200

0.189

0.019

46

none

synthetic

SF2

P41212

2.8

50.24

50.24

106.56

2,614

0.184

0.026

110

none

recombinant

NY5

P41212

2.3

50.09

50.09

107.39

4,111

0.194

O.oJ8

N(A'

70 unpublished resultsb

'"

HIV-l protease-inhibited complexes MVT-101

synthetic

SF2

P212121

2.3

51.7

59.2

62.45

7,943

0.176

0.019

64

�

acetyl-pepstatin

recombinant

NY5

P21212

2.0

58.39

86.70

46.27

12,901

0.176

O.oJ8

17

A-74704

recombinant

BHIO

P61

2.8

63.3

63.3

83.6

3,951

0.182

0.020

16

;;3

•

Information not provided in publication (N/A, not available).

bp.

M. D. Fitzgerald, B. M. McKeever, J. F. VanMiddlesworth, J. P. Springer.

-
t"' "C

'" a

.., tTl >'" tTl '"

w o '-0


3I 0

FITZGERALD

Figure 3

The structures of RSV

& SPRINGER

(left) and HIV-l (right) proteases.

Each structure is drawn

as a series of ribbon segments connecting the positions of a-carbon atoms. The structures

have been aligned so as to yield an overall rms deviation of 1.0 A; included in the alignment are indicated by shaded ribbon segments.

the 120 IX-carbon pairs

structural reports have appeared (19, 20). Weber and colleagues (107, 108) proposed a model for the structure of HIV-l protease, based on the structure of RSV protease. HIV-J Protease

Native HIV-I protease was crystallized (57) and its structure determined (46, 70, 110) simultaneously with the structural work on RSV protease. In



311

contrast to RSV protease, native HIV-I protease crystallizes as a sym metric dimer with one half of the dimer related to the other by a cry stallographic two-fold rotation. The overall topology of HIV-1 protease is similar to that of RSV protease as shown in Figure 3. All deletions in the smaller HIV-l protease (99 amino acids vs 124 amino acids for RSV protease) occur at surface loops so that 86 Q(-carbon atoms of an HIV- l monomer can be aligned with equivalent atoms in an RSV monomer with a rms deviation of 1.45 A (33). In contrast to RSV protease, the flaps in HIV-1 protease are ordered, adopting a position that is stabilized by intermolecular contacts. Comparisons with Structures of Native Monomeric Aspartyl Proteases

Overall, the structures of the two retroviral proteases align well with the structures of the monomeric aspartyl proteases. For instance, nearly half the Q(-carbon atoms of RSV protease can be aligned with the corresponding atoms in the monomeric aspartyl proteases with a rms deviation of 1.90 A or less (33). The similarity is strongest at the active site, such that 80 atoms in 22 amino acids of RSV protease can be superimposed on the corresponding atoms of rhizopuspepsin with a rms deviation of 0.45 A (33). Less closely related are the f3-sheets formed by the amino and carboxyl termini of the proteases; the six-stranded f3-sheet in the monomeric pro teases differs in orientation from the four-stranded f3-sheet in the retroviral proteases by about 50° (70). The most striking structural difference between the two classes of aspar tyl protease is that the retroviral proteases have two flaps, one in each monomer, instead of the single amino-terminal flap found in the mono meric aspartyl proteases. This difference may have implications for the mechanism of action of the two classes of enzymes, although the close structural homology in the active sites implies a common mechanism (11, 96). An additional difference is that all known retroviral proteases have an alanine three residues downstream from the active-site aspartic acid, while the monomeric aspartyl proteases typically have a serine or threonine in this position. This difference may correlate with the optimum pH for proteolysis (50, 91). Implications for Structures in Other Retroviral Classes

Because all of the known retroviral protease sequences can be fit into an alignment that consists of a conserved structural core and surface loops of varying lengths (105), their structures are likely very similar to RSV and HIV-l protease. This structural similarity is probably closer than for

312


the monomeric aspartyl proteases because the retroviral proteases are smaller and more conservative in their use of amino acids. THREE-DIMENSIONAL STRUCTURES OF INHIBITED COMPLEXES


HIV-J Protease

The crystal structures of HIV-I protease complexed with three different types of inhibitors (Figure 4) have been reported (16, 17, 64). A comparison

Figure 4

Structures of the compounds studied crystallographically in complex with HIV-I protease. (Upper) MVT-IOI, K, 780 nM (64). (Middle) Acetyl-pepstatin ( 17), K, 20 nM =

(80). (Lower) A-74704, K,

=

4.5

nM (16).

=



313

of the native enzyme structure and the structure of a complex with acetyl pepstatin shows that a core of 44 a-carbon atoms in each monomer can be aligned with a rms deviation of only 0.39 A (Figure 5). The remaining 55 amino acids show larger differences, the largest a 7-A difference in position between residues at the tip of the flap. Hydrophobic residues line the four central substrate-binding sites, in agreemcnt with the subsite specificities described above. Despite the fact that the crystal symmetry of each complex is different, the complexed structures appear to have similar conformations. For instance, the rms difference for all protein a-carbon positions between the MVT-I0l and A-74704 complexes is 0.65 A (16). The inhibitors all bind in extended conformations and form a similar pattern of hydrogen bonds and van der Waals contacts with both the body of the enzyme and the flaps (Figure 6). Another similarity among the three complex structures is a highly ordered water molecule found hydrogen bonded between each inhibitor and the flaps. No structures of inhibited complexes of RSV or AMV proteases have been reported, despite the fact that submicromolar inhibitors of these enzymes have been synthesized (93). It will be important to study com plexes from more than one enzyme to explore differences and similarities among the various retroviral proteases.

Figure 5

The structures of native HIV- I protease (solid lines) and the protein portion of the complex of HIV- I protease with acetyl-pepstatin (dashed lines) are drawn as lines connecting the positions of IX-carbon atoms. In this stereo pair, the IX-carbon positions of the amino acids used in the superpositions of the two structures (rms deviation 0.39 A for 88 IX-carbon pairs) are connected by thicker lines. =

314



G·248

Figure 6

G·248

The binding of acetyl·pepstatin to HIV-l protease. In this stereo pair, atoms in

residues 25-30, 48-50, 225-229, and 248-250 of the protease are drawn connected by thin lines; atoms in acetyl-pepstatin are connected by thicker lines. Dashed lines indicate potential hydrogen bonding interactions; black circles indicate oxygen atoms; and gray circles indicate nitrogen atoms.

Comparison with Structures of Inhibited Monomeric Aspartyl Proleases

The effect of binding of inhibitors appears to be much larger in the retro viral proteases than in the monomeric aspartyl protease primarily because of the large flap movement. However, it may not be accurate to assume that the differences observed between the native and inhibited structures of HIV-I protease represent structural changes induced by inhibitor binding because the flaps in the native structure may be in an overextended position stabilized by intermolecular contacts. During catalysis, the flaps are thought to perform a function similar to that of the single flap found in the monomeric aspartyl proteases, helping to bind the substrate for proteolysis and shielding the reaction from solution. In both cases, the residues in the flap form a fJ-ribbon with a tight turn at the tip. In the monomeric aspartyl-protease inhibitor complexes, this single /J-ribbon is oriented with its flat face towards the inhibitor (5, 6, 18, 32, 96), while in the retroviral protease complexes the two symmetric fJ-ribbons are both oriented edge on (16, 17, 64). Another feature not seen in the monomeric aspartyl-protease complexes is the tightly bound water molecule found in all three retroviral protease inhibitor complexes (Figure 6). This water molecule mediates several hydrogen bonds between the inhibitors and the flaps, and although it may or may not be important in helping to bind a substrate, it clearly has implications in the design of more potent inhibitors of HIV-1 protease.


315


MUTAGENESIS

Mutagenesis experiments were critical in establishing the role of retroviral proteases in the maturation of viral particles and in characterizing the proteases as members of the aspartyl-protease family. Deletion mutants in the protease-coding region of the MoMuLV genome (8, 36) resulted in the generation of progeny virions with low infectivity. Although these virions budded and were released from the infected cell, they contained gag and gag-pol polyproteins that had not been processed to yield mature structural proteins and enzymes. Similar deletion experiments in HIV-l showed that protease-deficient mutants produced noninfective, immature viral particles containing unprocessed polyproteins (74), and experiments with HTLV-I showed that the gag precursor accumulated when the pro tease coding region was deleted (69). Site-directed mutagenesis experiments showed that single-site mutation of the putative active-site Asp residue to Ala (48), Thr (88), and Asn (38) in HIV-1 and to Gly in HTLV-I (69) eliminated polyprotein processing, thus supporting the hypothesis that these enzymes are aspartyl proteases. Moreover, transfection of proviral DNA containing the D25N mutant into a human cell line resulted in the production of noninfectious progeny virions that contained unprocessed gag p55 (38). Single-site mutations have been used to probe the structure of the protease. Loeb et al (52, 53) examined 330 different randomly generated single-site missense mutations in HIV-l protease. They found that three noncontiguous regions in the enzyme (22-33, 47-52, and 74-87) are extremely sensitive to change. Figure 6 shows that residues in each of the first two of these regions participate in hydrogen bonding interactions with the inhibitor in the HIV-l protease complex structure; residues in the third region line the substrate-binding subsites. Louis et al (54) showed that modification of two highly conserved amino acid positions, Arg-87 and Arg-88 (R87K, R87E, and N88E), resulted in complete loss of proteolytic activity. Arg-87 participates in a network of interactions with two other highly conserved residues, Asp-29 and Arg-208, and Asn-88 is hydrogen bonded to hoth the amide nitrogen and the or atom of Thr-31, another highly conserved residue (106). Thus it is not surprising that mutations at these points should result in inactive protease. Nam et al (69) showed that a D93G mutant in HTLV-I protease eliminated polyprotein proces sing; why this change stops the processing, given the structural role of the analogous residues in RSV (Val-IOI ) and HIV- l (Val-77), is not obvious. The proximity of the amino terminus of one monomer to the carboxyl terminus of the second monomer has allowed the construction of a single-

3 16

FITZGERALD

& SPRINGER

polypeptide-chain form of HIV-I protease, in which two monomers have been linked by a Gly-Gly spacer (13). This construct allows the generation of protease molecules that are altered in only one half of the active site; mutation at a single catalytic aspartic acid (D126N and D126E) generated molecules that would not hydrolyze substrate.


ROLE OF SYMMETRY IN RETROVIRAL PROTEASE STRUCTURES Genetics

Although a high degree of structural homology clearly exists bctween the retroviral and the monomeric aspartyl proteases, the nature of the evolutionary relationship between the two groups of proteases is unclear. In 1978, Tang et al (97) postulated that the monomeric aspartyl proteases, which are composed of two pseudosymmetric domains, arose from an earlier gene duplication event. When researchers realized that the retroviral proteases were made up of two identical monomers, Pearl & Taylor (73) proposed that the proteases represented "fossil" examples of an ancestral dimeric protease from which the monomeric aspartyl proteases evolved. An alternative proposal is that through genetic recombination the retro viral genome assimilated a single domain of a monomeric aspartyl protease, and that when translated this single domain dimerized to form an active enzyme (34). Either hypothesis appears viable at this point. For instance, various open reading frames in vaccinia, pox, and [enti viruses code for proteins of unknown function that have been called pseudo proteases because of their sequence homology with the retroviral proteases (61, 76, 92), although the active site residues Asp-Thr(Ser)-Gly have not been retained. In addition, two identical N-terminal domains of porcine pepsin (a monomeric aspartyl protease) form an active aspartyl protease (2). However, other reasons may explain the symmetric nature of the retro viral proteases. Retroviruses may have to overcome the limitations of minimal genomes by using two identical monomers instead of a single two-domain protein to form an active enzyme. Also, because a retroviral protease is not active until the two monomers fold together, dimer for mation may be an important regulator of protease activity (59) and there fore virus maturation. As a group, the retroviral proteases do not appear to be as catalytically efficient as the monomeric aspartyl proteases. For example, kcat for HIV-l protease is 70 S-1 for the best known peptide substrates (59), while kcat is 400 S-1 for pepsin (82). The constraints imposed on retroviral proteases by their symmetrical dimeric nature may prevent


31 7

them from evolving into more efficient enzymes. Also, no force may exist to drive the retroviral proteases towards greater efficiency because the ratio of protease to target sites in virus replication appears to be as low as 1/6 to 1/60 (41).


Deviations from Absolute Symmetry

The role of absolute structural symmetry in enzyme catalysis and inhibition is of great interest. Although the native retroviral protease may be perfectly symmetric or inherently asymmetric from the beginning, when the enzyme binds an asymmetrical substrate or inhibitor, the complex is clearly asym metric. Significant local deviations still exist, even though the pseudo-C2 symmetric inhibitor A-74704 seems to bind in a more symmetric manner (1 6) than MVT-101 (64) or acetyl-pepstatin (17). These deviations may result from crystal packing forces, the intrinsic asymmetry of the dimer, or the small asymmetry of the inhibitor. Further high-resolution diffraction experiments should help clarify this point.

SUMMARY AND FUTURE DIRECTIONS

The need to develop safe and effective therapies for the treatment of AIDS has stimulated many groups to study the retroviral proteases, particularly the protease from HIV- l . Research reports appear seemingly daily, and a review of this nature is necessarily outdated before it is published. From a crystallographic perspective, a conservative estimate is that more than twenty structures of inhibited complexes of HIV-I protease have been examined to date, although only three have been published as of this writing. This enzyme will be studied by a larger number of independent investigators, in a greater variety of crystalline modifications, and with a greater variety of ligands than has ever been the case for an enzyme before. This wealth of structural information will provide an unprecedented opportunity for assessing the contributions of various protein-ligand inter actions (hydrogen bonds, van der Waals interactions, etc) to observed inhibition constants, for studying the structural plasticity of the enzyme itself, and for examining the effect of crystal packing forces on protein conformation.

ACKNOWLEDGMENTS

We are grateful to Dr. J. Erickson of Abbott Laboratories for sharing his results with us prior to publication.

318



Literature Cited 1. Benn, S., Rutledge, R., Folks, T., Gold, 1., Baker, L., et al. 1985. Science 230: 949 2. Bianchi, M., Boigegrain, R. A., Castro, B., Coletti-Previero, M.-A. 1990. Bio chem. Biophys. Res. Commun. 167: 339 3. Billich, S., Knoop, M.-T., Hansen, J., Strop, P., Sedlacek, J., et al. 1988. J. Bioi. Chern. 263: 17905 4. Blundell, T. L., Jenkins, J. A., Sewell, B. T., Pearl, L.

H., Cooper, J.

1990. J. Mol. Bioi. 211: 919

B., et al.

5. Bott, R., Subramanian, E., Davies, D. R. 1982. Biochemistry 21: 6956 6. Cooper, J. B., Foundling, S. I., Blun 7.

dell, T. L., Boger, J., Jupp, R. A., Kay, J. 1989. Biochemistry 28: 8596 Copeland, T. D., Oroszlan, S. 1988.

Genet. Anal. Technol. 5: 109 8. Crawford, S., Goff, S. P. 1985. J. Virol. 53: 899 9. Darke, P. L., Leu, C.-T., Davis, L. J.,

Heimbach, J. C, Diehl, R. E., et al.

1989. J. Bioi. Chem. 264: 2307 10. Darke, P. L., Nutt, R. F., Brady, S. F.,

Garsky, V. M., Ciccarone, T. M., et al.

1988. Biochem. Biophys. Res. Commun. 156: 297 11. Davies, D. R. 1990. Annu. Rev. Biophys. Biophys. Chem. 19: 189 12. Debouck, C., Gorniak, J. G., Strickler,

J. E., Meek, T. D., Metcalf, B. W.,

13.

Rosenberg, M. 1987. Proc. Natl. Acad. Sci. USA 84: 8903 Dilanni, C. L., Davis, L.

J.,

Holloway,

M. K., Herber, W. K., Darke, P. L., et

al. 1990. J. BioI. Chem. 265: 17348 14. Dittmar, K. J., Moelling, K. 1978. J. Virol. 28: 106

15. Dreyer, G. B., Metcalf, B. W., Tomaszek, T. A. Jr., Carr, T. J., Chandler, A. C. III, et al. 1989. Proc.

Natl. Acad. Sci. USA 86: 9752

16. Erickson, J., Neidhart, D. J., VanDrie, J., Kempf,

D.

J., Wang, X. c., et al.

1990. Science 249: 527

17. Fitzgerald, P. M. D., McKeever, B. M.,

VanMiddlesworth, J. F., Springer, J. P., Heimbach, J. c., et al. 1990. J. Bioi.

Chern. 265: 14209 18. Foundling, S. I., Cooper, J., Watson, F. E., Cleasby, A.,

Pearl, L. H.,

et al.

1987. Nature 327: 349

19. Foundling, S. I., Salemme, F. R.,

Korant, B., Wendoloski, J. J., Weber, P. C., et al. 1989. See Ref. 44, p. 181 20. Fraser, M., Hayakawa, K., Yoshinaka, Y., Katoh, I., James, M. N. G. 1989. See Ref. 44, p. 169 21. Giam, c.-Z., Boros, I. 1988. J. Bioi.

Chern. 263: 14617

22. Gilliland, G. L., Winborne, E. L., Nachman, J., Wlodawer, A. 1990. Pro teins 8: 82 23. Graves, M. c., Lim, J. J., Heimer, E. P., Kramer, R. A. 1988. Proc. Nat!. Acad. Sci. USA 85: 2449 24. Hansen, J., Billich, S., Schulze, T., Sukrow, S., Moe/ling, K. 1988. EMBO J. 7: 1785 25. Heimbach, J. c., Garsky, V. M.,

Michelson, S. R., Dixon, R. A. F., Sigal, I. S., Darke, P. L. 1989. Biochem.

Biophys. Res. Cornmun. 164: 955 26. Henderson, L. E., Benveniste, R. E.,

Sowder, R., Copeland, T. D., Schultz, A. M., Oroszlan, S. 1988. J. Viral. 62:

2587 27. Hostomsky, Z., Appelt, K., Ogden, R. C. 1989. Biochem. Biophys. Res. Com rnun. 161: 1056 28. Jacks, T., Power, M. D., Masiarz, F. 29.

R., Luciw, P. A., Barr, P. J., Varmus, H. E. 1988. Nature 331: 280 Jacks, T., Townsley, K., Varmus, H. E., Majors, J. 1987. Proc. Natl. Acad.

Sci. USA 84: 4298 30. Jacks, T., Varmus, H. E. 1985. Science 230: 1237 31. James, M. N. G., Sielecki, A. R. 1983. J. Mol. Bioi. 163: 299-361 32. James, M. N. G., Sielecki, A., Salituro, F., Rich, D. H., Hofmann, T. 1982. Proc. Natl. Acad. Sci. USA 79: 6137 33. Jaskolski, M., Miller, M., Mohana Rao, 1. K., Leis, J., Wlodawer, A. 1990. Biochemistry 29: 5889 34. Katoh, I., Ikawa, Y., Yoshinaka, Y. 1989. J. Virol. 63: 2226 35. Katoh, I., Yasunaga, T., Ikawa, Y., Yoshinaka, Y. 1987. Nature 329: 654 36. Katoh, I., Yoshinaka, Y., Rein, A.,

Shibuya, M., Odaka, T., Oroszlan, S.

1985. Virology 145: 280 37. Kay, J., Dunn, B. M. 1990. Biochem. Biophys. Acta 1048: I 38. Kohl, N. E., Emini, E. A., Schleif, W. A., Davis, L. J.,

Heimbach, J. c., et al.

1988. Proc. Natl. Acad. Sci. USA 85: 4686

39. Konvalinka, J., Strop, P., Velek, J., Cerna, V., Kostka, V., et al. 1990. FEBS Lett. 268: 35 40. Kotler, M., Danho, W., Katz, R. A., Leis, J., Skalka, A. M. 1989. J. Bioi. Chem. 264: 3428 41. Kotler, M., Katz, R. A., Danho, W., Leis, J., Skalka, A. M. 1988. Proc. Natl. Acad. Sci. USA 85: 4185 42. Kotler, M., Katz, R. A., Skalka, A. M. 1988. J. Viral. 62: 2696 43. Kriiusslieh, H.-G., Ingraham, R. H.,

RETROVIRAL PROTEASES Skoog, M . T., Wimmer, E., Pallai, P. V., Carter, C. A. 1 989. Proc. Nat!.

62.

44. Krausslich, H.-G., Oroszlan, S., Wim mer, E., eds. 1989. Viral Proteinases

63.

Acad. Sci. USA

86: 807

as Targets for Chemotherapy. Cold

Spring Harbor, NY: Cold Spring Har bor Laboratory Press. 287 pp. 45. Kriiusslich, H.-G., Schneider, H . , Zy barth, G., Carter, C. A., Wimmer, E.


1 98 8 .

46.

47.

J. Virol. 62: 4393

Lapatto, R., Blundell, T., Hemmings, A., Overington, J., Wilderspin, A., et al. 1989. Nature 342: 299 Le Grice, S. F. J., Ette, R., Mills, J., Mous, J . 1989. J. Bioi. Chem. 264: 14902

48. 49.

F. J., Mills , 1988. EMBO J . 7 : 2547

Le Grice, S.

Leis, J., Baltimore, D., Bishop, J. M . , Coffin, J., Fleissner, E . , e t al. 1988. J. Virol.

62: 1808

66.

55.

56.

68.

69. 70.

73.

57.

Louis, J. M., Wondrak, E. M., Cope land, T. D., Smith, C. A. D., Mora, P. T., Oroszlan, S. 1989. Biochem. Bio

76.

159: 87

75.

77.

78.

59.

McQuade, T. J., Tomasselli, A. G., Liu, L., Karacostas, V., Moss, B., et al. 80.

Meek, T. D., Dayton, B. D., Metcalf, B. W., Dreyer, G. B., Strickler, J. E., et al. 1989. Proc. Natl. Acad. Sci. USA

81.

61.

Meek, T. D., Lambert, D. M . , Dreyer, G. B., Carr, T. J., Tomaszek, T. A. Jr., et al. 1990. Nature 343: 90 Mercer, A . A., Fraser, K. M., Stockwell, P. A., Robinson, A. J. 1989. Virology 172:

665

Mous, J., Heimer, E. P., Le Grice, S .

F . J . 1988. J. Virol. 62: 1433

Muesing, M . A., Smith, D. H . , Cabra dilla, C. D., Benton, C. V., Lasky, L. A., Capon, D. J. 1 985. Nature 313: 450 Nam, S. H., Kidokoro, M., Shida, H . , Hatanaka, M . 1988. J . Virol. 62: 3718 Navia, M. A., Fitzgerald, P. M. D., McKeever, B. M., Leu, C.-T., Heim bach, J. c., et al. 1 989. Nature 337: Nutt, R. F., Brady, S. F., Darke, P. L., Ciccarone, T. M . , Dylion Colton, c., et al. 1 988. Proc. Natl. Acad. Sci. USA Pearl, L. H., Taylor, W. R.

1987.

Pearl, L. H., Taylor, W. R.

1987.

Nature 328:

482

Nature 329:

351

Peng, c., Ho, B. K., Chang, T. W., Chan g, N. T . 1989. J. Virol. 6 3 : 2550 Pichuantes, S . , Babe, L. M., Barr, P. J., Craik, C. S. 1989. Proteins 6: 324 Power, M. D., Marx, P. A., Bryant M . L . , Gardner, M . B . , Barr, P. J., Luciw, P. A. 1986. Science 231: 1567 Ratner, L., Haseltine, W., Patarca, R., Livak, K. J., Starcich, B., et al. 1985. ,

Nature 3 1 3:

277

Richards, A. D., Broadhurst, A. V., Ritchie, A. J., Dunn, B. M., Kay, J. FEBS Lett. 253: 214

Richards, A. D., Phylip, L. H . , Farmerie, W. G . , Scarhorough, P. E., Alvarez, A., et al. 1990. J. BioI. Chem. 265: 7733

1990. Science 247: 454

86: 1841 60.

159: 420

Moore, R., Dixon, M., Smith, R., Peters, G., Dickson, C. 1 987. J. Virol.

1989. 79.

1919

58.

phys. Res. Commun.

85: 7 1 29

72.

164: 30

Margolin, M . , Heath, W., Orborne, E., Lai, M., Vlahos, C. 1990. Biochem. Bio phys. Res. Commun. 1 67: 554 McKeever, B. M., Navia, M. A., Fitz gerald, P. M. D., Springer, J. P., Leu, C.-T., et al. 1989. J. Bioi. Chem. 264:

Miller, M . , Schneider, J., Sathy anarayana, B. K., Toth, M. V., Mar shall, G. R., et al. 1989. Science 246:

615

71.

74.

phys. Res. Commun.

Miller, M., Leis, J., Wlodawer, A. 1988.

J. Mol. Bioi. 204: 2 1 1

61: 480

67.

Louis, J. M., Smith, C. A. D., Wondrak, E. M., Mora, P. T . , Orosz Ian, S. 1989. Biochem. Biophys. Res. Commun.

Nature 337: 576

1 149

397

54.

Miller, M., Jaskolski, M . , Mohana Rao, J. K., Leis, J., Wlodawer, A. 1 989.

65. Moore, M . L., Bryan, W. M., Fak houry, S. A., Magaard, V. W., Huff man, W. F., et al. 1989. Biochem. Bio

J., Mous, J .

Leis, J., Bizub, D., Weber, I. T., Cameron, c., Katz, R., et al. 1989. See Ref. 44, p. 235 51. Lillehoj, E. P., Salazar, F. H. R., Mervis, R. J., Raum, M . G., Chan, H. W., et al. 1988. J. Virol. 62: 3053 52. Loeb, D. D., Hutchison, C. A. Ill, Edgell, M. H., Farmerie, W. G., Swanstrom, R. 1989. J. Virol. 6 3 : I I I 53. Loeb, D. D., Swanstrom, R., Everitt, L., Manchester, M., Stamper, S. E., Hutchison, C. A. III. 1989. Nature 340:

50.

64.

319

82. 83.

Richards, A. D., Roberts, R., Dunn, B. M., Graves, M . c., Kay, J. 1989. Ft;BS

Lett.

247: 1 13

Roberts, N. A., Martin, J. A., Kin chington, D., Broadhurst, A. V., Craig, J. C., et al. 1990. Science 248: 358 Sachdev, G. P., Fruton, 1. S. 1970. Bio chemistry 9:

4465

Sanchez-Pescador, R., Power, M. D., Barr, P. J., Steimer, K. S., Stempien, M. M . , et al. 1985. Science 227: 484


320


84. Sauer, R. T., Allen, D. W., Niall, H. D. 1981. Biochemistry 20: 3784 85. Schechter, I., Berger, A. 1967. Biochem. Biophys. Res. Commun. 27: 157 86. Schneider, J., Kent, S. B. H. 1988. Cell 54: 363 87. Schwartz, D. E., Tizard, R., Gilbert, W. 1983. Cell 32: 853 88. Seelmeier, S., Schmidt, H., Turk, V., von der Helm, K. 1988. Proc. Natl. Acad. Sci. USA 85: 6612 89. Sepu lveda P., Marciniszyn, J. Jr., Liu, D., Tang, J. 1975. J. Bioi. Chem. 250: 5082 90. Shinnick, T. M., Lerner, R. A., Sutcliffe, J. G. 1981. Nature 293: 543 91. Sielecki, A. R., Hayakawa, K., Fuji ,

naga, M., Murphy, M. E. P., Fraser, M., et al. 1989. Science 243: 1346 92. Slabaugh, M. B., Roseman, N. A. 1989.

Proc. Natl. Acad. Sci. USA 86: 4 152 93. Strop, P., Konvalinka, J., Pavlickova, L., Blaha , I., Soucek, M., et al. 1989. See Ref. 44, p. 259 94. Subramanian, E., Swan, I. D. A., Liu,

M., Davies, D. R.,

Jenkins,

J. A.,

et al.

1977. Proc. Natl. A cad. Sci. USA 74: 556

95. Suguna, K., Bott, R. R., Padlan, E. A., Subramanian, E., Sheriff, S., et al. 1987. J. Mol. BioI. 196: S77 96. Suguna, K., Padlan, E. A., Smith, C.

W., Carlson, W. D., Davies, D. R . 1 987 . Proc. Natl. A cad. Sci. USA 84:

7009 97. Tang, J., James, M.

N. G., Hsu, I. N., Jenkins, J. A. , Blundell, T. L. 1978.

Nature 271 : 6 18

98. Toh, H., Ono, M., Saigo, K., Miyata, T. 1985. Nature 3 15: 691

99. Tomasselli, A. G., Hui , J. 0., Sawyer,

T. K., Staples, D. J., FitzGerald, D. J., et al. 1990. J. Bioi. Chem. 265: 40S 100. Tomasselli, A. G., Olsen, M. K., Hui, J. 0., Staples, D. J., Sawyer, T. K., et

al. 1990. Biochemistry 29: 264 101. Umezawa, H., Aoyagi, T., Morishima,

H., Matsuzaki, M., Hamada, M., Takeuchi, T. 1970. J . Antibiot. 23:

259 102. Vogt, V. M., Wight, A., Eisenman, R. 1979. Virology 98: 154 103. von der Helm, K. 1977. Proc. Natl. Acad. Sci. USA 74: 9 11 104. Wain-Hobson, S., Sonigo, P., Danos, 0., Cole, S., Alizon, M. 1985. Cell 40: 9 105. Weber, T. T. 1 989. Gene 85: 565 106. Weber, I. T. 1990. J. Bioi. Chern. 265: 10492 107. Weber, I. T. 1990. Proteins 7: 172

l OS. Weber, I. T., M iller, M., Jaskolski, M., Leis, J., Skalka, A. M., Wlodawer, A.

1989. Science 243: 928 109. Wil son, W., Braddock, M., Adams, S.

E., R athjen, P. D., Kingsman, S. M., Kingsman, A. J. 1988. Cell 5 5 : 1 159

110. Wlodawer, A., Miller, M., Jaskolski,

M., Sathyanarayana, B. K., Baldwin, E., et al. 1989. Science 245: 616 111. Yoshinaka, Y., Katoh, I., Copeland, T. D., Oroszlan, S. 1985. J. Viral. 55:

870 1 12. Yoshinaka, Y., Katoh, I., Copeland, T. D., Oroszlan, S. 1985. Proc. Nat!. Acad. Sci. USA 82: 1618

1 1 3. Yoshinaka, Y., Katoh, I., Copeland, T. D., Smythers, G. W., Oroszlan, S. 1986. J. Virol. 57: 826

ADAM Proteases and Gastrointestinal Function.

Genome-wide identification and structure-function studies of proteases and protease inhibitors in Cicer arietinum (chickpea).

Serine proteases: structure and mechanism of catalysis.

Retroviral Integrase Structure and DNA Recombination Mechanism.

On the nucleotide composition and structure of retroviral RNA genomes.

Characterization and function of intracellular proteases in sporulating Bacillus cereus.

Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer.

Proteases and small intestinal barrier function in health and disease.

Viroids: structure and function.

Phycobilisome structure and function.

Histone structure and function.

Parkin structure and function.

Nucleosome structure and function.

Osteoclasts: structure and function.

Structure and function of telomeres.

αIIbβ3: structure and function.

Structure and function of profilin.

Extracellular proteases of Aspergillus flavus. Fungal keratitis, proteases, and pathogenesis.

Phosphorylation-Dependent Activation of the ESCRT Function of ALIX in Cytokinetic Abscission and Retroviral Budding.

Function, therapeutic potential and cell biology of BACE proteases: current status and future prospects.

Ibuprofen regulates the expression and function of membrane-associated serine proteases prostasin and matriptase.

Function and clinical relevance of kallikrein-related peptidases and other serine proteases in gynecological cancers.

The structure and function of acid proteases. V. Comparative studies on the specific inhibition of acid proteases by diazoacetyl-DL-norleucine methyl ester, 1,2-epoxy-3-(p-nitrophenoxy) propane and pepstatin.

Inhibitors of Serine Proteases in Regulating the Production and Function of Neutrophil Extracellular Traps.