Mutation of Asn-475 in the Venezuelan Equine Encephalitis Virus

Subscriber access provided by READING UNIV

Article

Mutation of Asn-475 in the Venezuelan Equine Encephalitis Virus nsP2 Cysteine Protease leads to a Self-inhibited state Jaimee Compton, Matthew Mickey, Xin Hu, Juan J Marugan, and Patricia Marie Legler Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00746 • Publication Date (Web): 24 Oct 2017 Downloaded from http://pubs.acs.org on October 29, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Mutation of Asn-475 in the Venezuelan Equine Encephalitis Virus nsP2 Cysteine Protease leads to a Self-inhibited State Jaimee R. Compton†, Matthew Mickey§, Xin Hu‡, Juan J. Marugan‡, Patricia M. Legler*† †

U.S. Naval Research Laboratory, 4555 Overlook Ave., Washington, D.C., §U.S. Naval

Academy, Annapolis, MD, ‡National Center for Advancing Translational Sciences, NIH, Rockville, MD

AUTHOR EMAIL ADDRESS. [email protected] RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to) TITLE RUNNING HEAD. Self-inhibition of the VEEV nsP2 Cysteine Protease. CORRESPONDING AUTHOR FOOTNOTE. 202-404-6037 (telephone)

ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT. The alphaviral nsP2 cysteine protease of the Venezuelan equine encephalitis virus (VEEV) is a validated anti-viral drug target. Clan CN proteases contain a cysteine protease domain that is intimately packed with an S-adenosyl-L-methionine dependent RNA methyltransferase (SAM MTase) domain. Within a cleft formed at the interface of these two domains, the peptide substrate is thought to bind. The nucleophilic cysteine can be found within a conserved motif,

475

NVCWAK480, which differs from that of papain (22CGSCWAFS29).

Mutation of the motif residue, N475, to alanine unexpectedly produced a self-inhibited state where the N-terminal residues flipped into the substrate-binding cleft. Notably, the N-terminal segment was not hydrolyzed, consistent with a catalytically incompetent state. The N475A mutation resulted in a 70-fold decrease in the kcat/Km. A side-chain to substrate interaction was predicted by the structure; the S701A mutation led to a 17-fold increase in the Km. An Asn at the n-2 position relative to the Cys was also found in the coronaviral papain-like proteases/deubiquitinases (PLpro) of the SARS and MERS viruses, and in several papain-like human ubiquitin specific proteases (USP). The large conformational change in the N475A variant suggests that Asn-475 plays an important role in stabilizing the N-terminal residues and in orienting the carbonyl during nucleophilic attack, but does not directly hydrogen bond the oxyanion. The state trapped in crystallo is an unusual result of site-directed mutagenesis, but reveals the role of this highly conserved Asn and identifies key substrate-binding contacts that may be exploited by peptide-like inhibitors.

KEYWORDS. Alphavirus, VEEV, nsP2, cysteine protease, self-inhibited, flipped, kinetics, structure


Page 2 of 33

Page 3 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

INTRODUCTION The Venezuelan equine encephalitis virus (VEEV) is a New World alphavirus that is typically spread by mosquitos; it is a member of the Togaviridae family. The alphaviral genome encodes structural and nonstructural proteins. Alphaviruses are Group IV (+)ssRNA viruses, their genomes are essentially messenger RNAs. Translation occurs early after entry into the cell and precedes replication of the genome. Translation of the (+)ssRNA genome produces a single polypeptide containing the nonstructural proteins (nsP), nsP123 or nsP1234, if read through of an opal stop codon occurs (1). Polyprotein processing is an essential step during viral replication and proteolytic cleavage occurs in a specific order. The nsP2pro cleavage site motifs are found in between the nsP, and cleavage produces nsP1, nsP2, nsP3, and nsP4 (Figure 1). The nsP2 cysteine protease (nsP2pro, EC 3.4.22.-) plays an important role in regulating the replication of the RNA genome (2).

The nsP123 cleavage intermediate and the RNA-dependent RNA

polymerase of nsP4 synthesizes minus strand copies of the genome, while synthesis of the plus strand requires complete processing of the polyprotein by the nsP2pro (3;4). Processing intermediates, nsP23 and nsP34, are also detectable early in infection (5;6). Proteolytic cleavage of the nsP23 intermediate in trans triggers a template switch, shifting production from minus strand copies of the viral genome to the production of positive strand copies of the (+)ssRNA viral genome (7). The positive sense strands are then packaged into virions. Alphaviral nsP2's are multi-domained proteins. Nonstructural protein 2 contains: an Nterminal region of unknown function, a helicase, a cysteine protease, and an S-adenosyl-Lmethionine dependent RNA methyltransferase (SAM MTase) domain (Figure 1). The MTase has not been shown to be active to date, but is thought to carry a nuclear localization signal (8).


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 33

Figure 1. A

nsP1

nsP2

nsP3

nsP4

TREEFEAFVAQQQRFDAGA↓YIFSSD VEEPTLEADVDLMLQEAGA↓GSVETP LSSTLTNIYTGSRLHEAGC↓APSYHV

B

C Residues 473-477 A' B' 471-477

D

E

6 5 4 3 2 1

VEEV nsP12 VEEV nsP23 VEEV nsP34 N-terminal

↓1'2'3'4'5'6'

VEEPTLEADVDLMLQEAGA↓GSVETP LSSTLTNIYTGSRLHEAGC↓APSYHV TREEFEAFVAQQQRFDAGA↓YIFSSD 471 QNKAAV C477

Pseudo-Substrate

Figure 1. Mimicry of the substrate-bound state.

(A) Organization of the nonstructural

polyprotein and locations of intervening nsP2 protease cleavage sites. (B) The structure of the


Page 5 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

free enzyme (PDB ID 2HWK) is shown on the left. In white is the cysteine protease domain, and in green is the SAM methyltransferase domain. In slate blue is the nucleophilic Cys-477. In red is the β-hairpin; the substrate is thought to bind at the interface of the two domains beneath the β-hairpin. The N475A mutation led to a dramatic conformational change in the N-terminal residues. On the right is the structure of the N475A variant (PDB ID 6BCM). The two conformations of the N-termini (A’ and B’) are shown in ball and stick. The B' conformer was the predominant conformation. (C) Cartoon depiction of the conformational change. (D) The Nterminal sequence mimics the cleavage site motif sequence; however, no cleavage occurred consistent with a catalytically incompetent state. In the N475A variant, Ala-475 occupied the S2 subsite, and hydrogen bonded to the backbone NH of His-510. (E) A hydrogen bond between His-546 and the backbone of Val-476 in the S1 subsite pulled the loop closer to the substrate.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The alphaviral nsP2 cysteine proteases are validated drug targets that have been demonstrated to be essential for viral replication by genetic methods (9-12). Deletion of the Cterminal half of the nsP2 (i.e. the cysteine protease and SAM MTase domains) abolishes proteolytic processing of the nonstructural polypeptide (13;14). The nucleophilic Cys found in the conserved

475

NVCWAK480 motif has been conclusively identified; Stauss et al.(15) and

Merits et al. (16;17) have shown that mutation of the Cys results in the loss of protease activity and abolishes the production of infectious RNA from a cDNA clone. Covalent inhibition of the nsP2pro by E64d also confirmed the assignment (PDB 5EZS (18)). For in vitro assays and the determination of inhibitor-bound states by X-ray crystallography, nsP2 constructs lacking the Nterminal region and the helicase (residues 1-456) have been successfully expressed and purified from E.coli in high yield (19). Proteolytic activity can be readily detected in vitro from VEEV and CHIKV constructs containing the nsP2 cysteine protease and SAM MTase using continuous and discontinuous assays (20-22). No other intracellular mammalian proteases have been shown to cleave the nonstructural polypeptide (23-25). While key substrate binding interactions would be valuable to exploit for small molecule inhibitor development, the substrate-bound state of an alphaviral protease has eluded structure determination. To date at least two models of the bound substrate have been published (26;27). Ideally small molecule anti-viral inhibitors should engage highly conserved residues with indispensable functions. The conserved motif residues are important to catalysis, active site structure, product, and substrate binding.

To identify residues required for catalysis and

substrate binding we previously mutated seven residues: N475, K480, N545, R662, K705, K706, and P713 (28). 475

N475 which is conserved in the alphaviral nsP2 cysteine protease motif,

NVCWAK480, is also conserved in the papain-like coronaviral protease motif (29). The side-


Page 6 of 33

Page 7 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

chain (and backbone NH) of Asn-475 was found directed away from the carbonyl oxygen of the peptide-like inhibitor E64d (PDB 5EZS); however, mutation of N475A led to a significant decrease in the kcat/Km (24-fold in phosphate buffer at pH 6.0). No hydrogen bond between the side-chain of Asn-475 and the peptide-like inhibitor was observed. In our substrate-bound models, the side-chain of Asn-475 was either hydrogen bonded to Arg-662 of the MTase or directed towards the substrate. While site-directed mutagenesis is a well-established method for identifying residues important for substrate binding or catalysis the effects of mutations can in some cases be unpredictable. A decrease in the kcat/Km could imply that Asn-475 is important for binding and positioning the substrate in the active site for nucleophilic attack; however, here we found that the mutation of Asn-475 to alanine unexpectedly led to a large conformational change in the residues N-terminal to the nucleophilic Cys. The residues N-terminal to the nucleophilic Cys are important in papain and papain-like proteases; they often play a role in stabilizing the oxyanion (e.g. Gln-19 precedes Cys-25 in papain (30), Asn-271 precedes Cys-276 in ubiquitinspecific protease 2 (USP2) (31)). We discuss the structure of the self-inhibited N475A variant and the conservation of the hydrogen bonding interactions in other cysteine proteases.

MATERIALS AND METHODS Protein Expression and Purification The construction of the pet32a plasmid expressing residues 457-792 of the VEEV nsP2 was previously described (32). The N475A mutation was incorporated using a QuikChange kit (Agilent Technologies, Inc., Santa Clara, CA), and the mutation was confirmed by DNA sequencing. Protein was expressed and purified according to our previously published procedure


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(33).

Page 8 of 33

Protein was further purified using a G-200 Superdex gel filtration column (G.E.

Healthcare, Inc.) equilibrated with 50 mM Tris pH 7.6, 150 mM NaCl, 5 mM DTT. Enzyme Assays Wild type (WT) and mutant N475A and S701A VEEV nsP2 cysteine proteases were assayed using a cyan- and yellow-fluorescent protein (CFP-YFP) substrate containing 25 residues of the nsP1-nsP2 junction. The cleavage of the FRET substrate was monitored, and steady state kinetic parameters were determined as previously described (34;35).

Assays were run in 50 mM

HEPES or 100 mM sodium phosphate buffer pH 7.0 at room temperature 23 ± 3 °C. Data for the N475A variant was collected over a period of 140-240 min using 6.4-14 µM enzyme. For N475A calculations, the emission ratio obtained from the first point collected was used as the initial value of the uncut substrate. All data were collected in triplicate. Secondary Structure Analysis Thermal denaturation (2 °C/min) was monitored using circular dichroism (CD) spectroscopy and a Jasco 810 spectropolarimeter fitted with a Peltier temperature controller. Proteins (~0.2 mg/mL) were buffer exchanged into 1x PBS pH 7.4 using PD-10 columns. Data were collected at 222 nm. The apparent melting temperature (Tm) was determined from a four parameter fit of the data. Three melts were performed, the Tm values were averaged, and standard deviations were calculated. Data Collection Diffraction data were collected using Cu Kα radiation at 100 K with a Bruker D8 VENTURE consisting of an IµS 3.0 source, HELIOS MX optics and a PHOTON II CMOS detector. Crystals were grown in 0.1 M Bicine pH 8.5, 20% PEG 6000, 10% glycerol. Crystals were cryo-


Page 9 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

protected in precipitant containing 30% glycerol. Data were processed using the Bruker Proteum software suite. The structure was determined by molecular replacement using PDB 2HWK (36) and Phaser (37). Simulated annealing was performed using CNS 1.1, and the model was refined using REFMAC5 (38) and Phenix (39). Model building was performed using Coot (40).

RESULTS X-ray Crystal Structure of the N475A VEEV nsP2 Cysteine Protease. Alphaviral nsP2 cysteine proteases (nsP2pro) recognize a conserved cleavage site motif (Q/H/F)(E/D)AG(A/C)↓(G/A/Y) in their substrates. Unexpectedly, the mutation of N475 to alanine produced a sequence similar to the protease cleavage site motif, and the N-terminus of the protease was found in two alternate conformations (Figure 1B, 1C). The data collection and refinement statistics of the VEEV nsP2pro N475A structure are shown in Table 1. The A' conformer was directed out towards solvent, while the B' conformer was found flipped into the predicted binding site of the substrate (Figure 1). We previously showed that the same site was occupied by the peptide-like epoxysuccinyl inhibitor, E64d (PDB 5EZS, (41)) (Figure 2).

In

the structure of the self-inhibited N475A variant (PDB 6BCM), the five N-terminal residues 472

NKAAVC477 which contain the mutated N475A residue appear to occupy the predicted S1-S5

subsites (Schechter and Berger nomenclature (42)). The residues acted as a pseudo-substrate occupying a region that is thought to recognize the conserved cleavage site motif residues of the substrate Q(E/D)AG(C/A)↓(GAY) (Figure 1D).


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

Page 10 of 33

B

N475

D507 R662

C

D

N475A

Figure 2. Comparison of (A) the E64d-inhibited VEEV nsP2 cysteine protease (PDB ID 5EZS) with that of (B) the N475A variant (PDB ID 6BCM) shows that the N-terminus bound in the predicted substrate binding site beneath the β-hairpin. The 2Fo-Fc map is shown in blue at a


Page 11 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

sigma level of 1.0. Negative and positive density in the Fo-Fc was not visible at a sigma level of ±3.5. The inset shows the hydrogen bonding network between Asp-507, Asn-475, and Arg-662. (C) Alternate view of the hydrogen bonding interactions observed in the E64d-inhibited enzyme, and (D) in the N475A variant.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table 1. X-ray crystallography data collection and refinement statistics.

VEEV nsP2 N475A (PDB 6BCM) Space Group Unit Cell Dimensions a, b, c (Å) α, β, γ (°) Wavelength (Å) Resolution Range (Å) Unique Reflections Rsym I/σI Completeness Redundancy Refinement Statistics Resolution (Å) No. of Reflections Rfactor Rfree Number of Atoms Protein Solvent Other Average B-factors (Å) Protein Solvent R.m.s.d. from ideal geometry Bond lengths (Å) Bond angles (degrees) Ramachandran plot Most favored region (%) Additional allowed regions (%) Generously allowed regions (%) Disallowed regions (%) a

P1 42.53, 45.54, 47.77 78.3, 65.3, 88.8 1.54 44.46-2.08 (2.18-2.08) 18,603 (2,445) 0.081 (0.337) 9.73 (3.01) 97.4 % (97.0%) 3.29 (2.56)

44.46 (2.1) 15,941 0.198 0.243 2592 111 13 22.8 28.1 0.012 1.427 90.4% 9.2% 0.4% 0.0%

Values in parentheses are for the outer most data shell Rfree for test set and size of test set as % total reflections in parentheses.

b


Page 12 of 33

Page 13 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Despite the substitution of the negatively charged P4 residue with a Lys, the Lys-473 side chain was directed towards Lys-706 and Lys-705 and was 7.2 to 8.5 Å away. The orientation of the P4 residue correlated well with the two prior models of the substrate-bound state (43;44). The S4 subsite is thought to be formed by Lys-705 and Lys-706, two residues that belong to the SAM MTase domain and may recognize the Glu/Asp in the substrate (45-47). Lys-705 and Lys706 were shown to be important to binding by site-directed mutagenesis (48); mutation of both lysines led to a 4-fold increase in the Km (49). Notably, the self-inhibited state (alternate conformer B') was not autocatalytically cleaved in cis, and continuous density could be seen for residues 472-477. Similarly, no cleavage of the lower occupancy (occupancy = 0.40) A' conformer was observed, indicating that cleavage in trans did not occur at a detectable level. Residues 471-477 in the B' conformer could be refined with an occupancy of 0.60 without the appearance of negative density (-3.5σ level) in the Fo-Fc maps, suggesting that the self-inhibited bound state was the predominant state in the crystal (Figure 2B). The catalytic dyad is formed by Cys-477 and His-546. The lack of autocatalytic cleavage is consistent with the prior assignment of the backbone Cys-477-NH as the hydrogen bond donor to the oxyanion in the transition state (50;51). The rotamer of the His-546 side chain was well stabilized by a hydrogen bond between His-546-ND1 and the backbone carbonyl oxygen of Asn-544 (2.9 Å) in the β-hairpin (Figure 2D). The alphaviral nsP2pro cysteine proteases are thought to have catalytic dyads, rather than catalytic triads (52;53). The h-bond interaction between Asn-544 and His-546 may partially fulfil the role of the absent third residue (Suppl. Fig. S2). Movement of the β-hairpin loop was also observed in the N475A structure (Figure 1E). The loop moved upwards and was pulled closer to the pseudo-substrate by two h-bond


13

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 33

interactions (Suppl. Fig. S1), a backbone-to-backbone contact between Trp-547-NH to Val-476O and Asn-545-O to Val-476-NH. Val-476 appears to occupy the S1 subsite, and its side-chain was directed away from the interior of the cleft but was in close proximity to the His of the dyad (His-546-NE2 was 3.2 Å from the Val-476-CG2). The S2 subsite is the primary determinant of substrate specificity in cysteine proteases (54), while the S1 subsite is more important in serine proteases (55). A Gly residue is typically found at the P2 position of the substrate; in the pseudo-substrate an Ala occupied this position, and the side chain was directed towards a shallow hydrophobic pocket formed by Ile-542 and Trp-547. The side-chain conformations of residues within the cleft were largely unperturbed and were similar to those found in the free enzyme (PDB 5EZQ) and the E64d-inhibited enzyme (PDB 5EZS) (56). Several key contacts between the pseudo-substrate and residues within the substrate binding cleft were present (Figure 2). Backbone-to-backbone contacts were observed at four locations: His-510-O and Ala-475-NH (3.0 Å); His-510-NH and Ala-475-O (3.0 Å); Asn-545-O and Val-476-NH (3.0 Å); and Trp547-NH to Val-476-O (2.7 Å). The His-510-NH to Ala-475-O hydrogen bond was not mimicked in the peptide-like E64d inhibited enzyme where the directionality of the bound inhibitor (N to C) is reversed (Figure 2). This may account for the weak binding of the E64d inhibitor (Kp = 40 ± 10 µM) to the VEEV nsP2 cysteine protease. In other cysteine proteases E64d is a potent nanomolar inhibitor (57).

One side-chain to

substrate contact was observed between Asn-472-O (pseudo-substrate) and Ser-701-Oγ (enzyme). A second side-chain contact is possible between Asn-554-OD1 and Gln-471-NE2 (3.4 Å), however the density was weak for Gln-471. A summary of the contacts is shown in Figure 2. Effects of the S701A mutation on Steady State Kinetic Parameters.


14

Page 15 of 33

To determine if the bound conformation truly mimicked the substrate-bound state, we mutated S701 to alanine. Ser-701 interacted with Asn-472 in the pseudo-substrate. Asn-472 corresponds to the P5 residue of the substrate. Consistent with the bound state of the pseudosubstrate, the Km of the S701A variant increased significantly and was 17-fold higher than that of the WT enzyme (Table 2, Figure 3). The kcat also increased slightly (2.6-fold) consistent with weaker binding of the product and faster product release. Interestingly, both the CHIKV and VEEV nsP2 cysteine proteases can cleave a common substrate, namely the Old World Semliki Forrest virus (SFV) nsP1/nsP2 cleavage site (58;59). In New World alphaviruses the Ser-701 is invariant; however, in Old World alphaviruses this residue is substituted by a Lys or Gln. At least one of the P5 residues of SFV and VEEV are identical, while none of the P5 residues are identical to those of CHIKV. However, the nsP2pro of CHIKV and SFV tolerate the same P4 residue. Thus, the overlap in substrate specificity might be difficult to predict from sequence alone. 4000 WT

N475A

S701A

0.02

1 / v (U/mg)

0.03

S.A. (U/mg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

2000

0.01

S701A N475A

0 0

Figure 3.

20

40

60 80 100 [V12] µM

120

140

WT 0 -0.1

0 0.1 1 / [V12] µM

0.2

Effects of the S701A and N475A mutations on the Km and Vmax. The interaction

between the substrate and S701 was predicted from the self-inhibited N475A structure. Kinetic parameters were measured using 50 mM HEPES pH 7.0 buffer at room temperature and a CFPYFP substrate containing the VEEV nsP1/nsP2 cleavage site sequence.


15

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 16 of 33

Table 2. Steady state kinetic parameters collected at room temperature (23 ± 3 °C) using the CFP-YFP substrate containing VEEV nsP1/nsP2 cleavage site. 1

Values

Enzyme

were

previously

reported

in

ref.

(60)

Vmax (mU/mg)

kcat min-1

Km µM

kcat/Km 1/min*µ µM

Fold Change in kcat

Fold Change in Km

Fold Change in kcat/Km

WT1

135 ± 8

5.2 ± 0.3

110 ± 10

0.047 ± 0.005

---

---

---

100 mM NaPi pH 6.0

N475A1

20 ± 10

0.8 ± 0.4

400 ± 200

0.002 ± 0.001

7-fold

4-fold

24-fold

100 mM NaPi pH 6.0

WT

34 ± 1

1.30 ± 0.04

19 ± 3

0.07 ± 0.01

---

---

---

50 mM HEPES pH 7.0

N475A

0.60 ± 0.02

0.023 ± 0.001

24 ± 3

0.0010 ± 0.0001

57-fold

1.3-fold

70-fold

50 mM HEPES pH 7.0

S701A

90 ± 30

3±1

330 ± 160

0.009 ± 0.005

2.6-fold

17-fold

8-fold

50 mM HEPES pH 7.0

WT

36 ± 2

1.36 ± 0.08

270 ± 20

0.0050 ± 0.0005

---

---

---

100 mM NaPi pH 7.0

0.60 ± 0.06

0.023 ± 0.002

340 ± 40

0.00007 ± 0.00001

60-fold

1.3-fold

74-fold

N475A


Buffer

100 mM NaPi pH 7.0

Page 17 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Effects of the N475A mutation on Steady State Kinetic Parameters. We had previously measured the Km and kcat values in phosphate buffer near the pH optimum of the enzyme (61). Here we remeasured these parameters and obtained a lower Km in 50 mM HEPES pH 7.0 buffer. Asn-475 is conserved in alphaviral nsP2 cysteine proteases and is part of the conserved motif surrounding the nucleophilic Cys, 475NVCWAK480. The Asn is also conserved in papain-like proteases from coronaviruses, but is notably absent from the papain motif,

22

CGSCWAFS29. In crystal structures of the free enzyme (PDB 2HWK, 5EZQ) and

inhibitor-bound enzyme (PDB 5EZS), the backbone carbonyl of Asn-475 is within hydrogen bonding distance of Trp-478-Nɛ1 (3.1 Å) of the motif (62). The Trp of the motif has also been shown to be critically important in the Old World alphaviral CHIKV and SFV nsP2 proteases, and mutation to alanine abolished viral replication (63). This residue is also found in the papain motif. The Asn-475 side chain ND2 is hydrogen bonded to the backbone carbonyl of Asp-507, and the side chain of Asp-507 is hydrogen bonded to the backbone NH of Asn-475 in a reciprocating fashion (Figure 2A). From inspection of the structure, Asp-475 appears to make key contacts that stabilize the three dimensional fold and active site.

Addition of the N475A

variant to the substrate in our in vitro assays increased the emission ratio of the substrate consistent with substrate binding to the enzyme. The Km of the N475A variant was comparable to that measured for the WT enzyme under the same conditions (Figure 3, Table 2). While substrate binding was detectable, the mutant showed a 70-fold decrease in the kcat/Km suggesting that it is catalytically incompetent and that Cys-477 is poorly positioned to catalyze the reaction. The N475A mutation did not produce a strong competitive inhibitory effect on the Km in 50 mM HEPES buffer or in 0.1 M sodium phosphate buffer at pH 7 buffer; however, near the pH


17

Biochemistry

optimum of the enzyme the Km of the N475A variant in 0.1 M sodium phosphate pH 6 buffer increased and was 3.6-fold higher than that of WT (KmWT = 110 µM; KmN475A = 400 µM) (64). At pH 7 the Km of the WT enzyme and N475A variant was 14-fold higher in the phosphate buffer, than in the 50 mM HEPES pH 7 buffer. Effect of the N475A mutation on Melting Temperature (Tm) Circular dichroism (CD) spectroscopy was used to examine the effects of the mutations on protein stability (65). The melting temperatures of the N545A, S701A, K706Q, and P713S were comparable to that of WT and only differed by less than one degree. The yield of the S701A variant was significantly lower than the other variants. The Tm of the N475A variant was 2.1 degrees higher than that of the WT enzyme consistent with additional hydrogen bonding interactions in solution. Interestingly, the R662K variant produced a biphasic denaturation curve (Figure 4). 1.2 1

Fractional Ellipticity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 33

WT 0.8

R662K

N545A

0.6

S701A

0.4

K706Q

0.2

P713S

N475A

0 20

30

40

50

60

70

80

90

Temperature (C) Figure 4. Thermal denaturation of the WT VEEV nsP2pro and its variants in 1x PBS pH 7.4. Protein denaturation was monitored using CD spectroscopy. Data have been normalized and fractional ellipiticity is shown. Tm values are reported in the Suppl. Information.


18

Page 19 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

One key difference among the New and Old World nsP2 cysteine proteases is the residue homologous to Arg-662. In the VEEV nsP2 cysteine protease, Arg-662 is within hydrogen bonding distance of the Asn-475 side chain, while in the CHIKV, WEEV, and EEEV proteases the Arg-662 residue is substituted by an Asn, Asp, or Glu, respectively. While the side-chain conformation of Arg-662 was not significantly changed by the N475A mutation (Figure 2), the biphasic behavior of the R662K variant suggests that R662 may also stabilize the complex between the nsP2 protease and SAM MTase domains. Structural Similarity to Papain & Ubiquitin Hydrolases The alphaviral nsP2 cysteine proteases were initially categorized as papain-like Clan CA proteases (66;67), but in 2006 were later segregated into Clan CN. Both papain and the VEEV nsP2pro have their nucleophilic Cys at the N-terminal end of an alpha helix and the His of the dyad within a β-strand (68) (Suppl. Fig. S2). The nsP2 proteases are papain-like in their ordering of their Cys/His dyad residues (e.g. papain uses Cys-25/His-159 and VEEV nsP2 Cys477/His-546) and in their organization. From overlays of papain and the alphaviral nsP2pro 3 helical regions, 2 β-strands, and the catalytic dyad residues can be overlaid (Suppl. Fig. S2). The loop in between the two β-strands varies in length among structurally similar cysteine proteases (69). Beneath this loop is the substrate binding site for both enzymes. The Asn-475 residue in the alphaviral nsP2pro is also found in the coronaviral papain-like proteases (Figure 5). The similarities suggest a distant evolutionary relationship to papain, but also differences in mechanism.


19

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

A

Papain

VEEV

Page 20 of 33

SARS

USP5

B VEEV WEEV EEEV Sinbis CHIKV SFV Middleburg Aura Whataroa Tai Forest Eilat Ockelbo Mayaro Getah

Asn-475 Cys-477 Asp-507 DVFQNKANVCWAKALVPVLKTAGIDMTTEQWNTVD-YFETDKAHS DVFQNKVNVCWAKALEPVLATANIVLTRQQWETLH-PFKHDRAYS DVFQNKVNVCWAKALEPVLATANITLTRSQWETIP-AFKDDKAYS DPFSCKTNVCWAKALEPILATAGISLTGCQWSELFPQFADDKPHS DTFQNKANVCWAKSLVPILETAGIKLNDRQWSQIIQAFKEDKAYS DAFQNKANVCWAKSLVPVLDTAGIRLTAEEWSTIITAFKEDRAYS DPFQNKANVCWAKALKPVLATAGIRLSAAEWSDLIVAFKEDKAYS DPFASKVNTCWAKAIIPILRTAGIELTFEQWEDLFPQFRNDQPYS NPFSCKTNVCWAKALEPILSTAGISLTGCQWADLFPQFEDDKPHS NAFSCKTNVCWAKALVPILATAGITLTGAQWAELFPQFDRDEPHS NAFSCKTNVCWAKALVPVLATAGLKLSGAQWTELFPQFERDEPHS NPFSCKTNVCWAKALEPILATAGIVLTGCQWSELFPQFADDKPHS DVFQNKAKVCWAKCLVPVLETAGIKLSATDWSAIILAFKEDRAYS DPFQNKAKVCWAKCLVQVLETAGIRMTADEWNTI-LAFREDRAYS

Clan CN CN CN CN CN CN CN CN CN CN CN CN CN CN

SARS PLpro MERS PLpro TGEV MEV

TSIKWADNNCYLSSVLLALQQLEVKFNAPALQEAYYRARAGDAAN RSLKLSDNNCYLNAVIMTLDLLKDIKFVIPALQHAFMKHKGGDST IILKQGDNNCWINACCYQLQAFDFFNNEAWEKFKKGDVMDFVNLC FAFKQSNNNCYINVACLMLQHLSLKFPKWQWQEAWNEFRSGKPLR

PA PA ---

USP5 USP12 USP21 USP2 USP48 USP46 USP8

GIRN-LGNSCYLNSVVQVLFSIPDFQRKYVDKLEKIFQNAPTDPT GLVN-FGNTCYCNSVLQALYFCRPFREKVLAYKSQPRKKESLLTC GLRN-LGNTCFLNAVLQCLSSTRPLRDFCLRRDFRQEVPGGGRAQ GLRN-LGNTCFMNSILQCLSNTRELRDYCLQRLYMRDLHHGSNAH GLVN-FGNTCYCNSVLQALYFCRPFRENVLAYKAQQKKKENLLTC GLVN-FGNTCYVNSVLQALYFCRPFRENVLAYKAQQKKKENLLTC GLRN-LGNTCYMNSILQCLCNAPHLADYFNRNCYQDDINRSNLLG

CA CA CA CA CA CA CA

C.difficile Cwp84

KNQGSL-NTCWSFSGMSTLEAYLKLKGYGTYDLSEEHLRWWATGG

--


20

Page 21 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 5. Conservation of the motif Asn in other cysteine proteases. (A) A conserved Asn is found in the n-2 position relative to the Cys in the alphaviral nsP2 proteases, the papainlike proteases of SARS and MERS, and in ubiquitin specific proteases (USP), but not in papain. Structural overlays show that an Asn similar to Asn-475 in the VEEV nsP2 cysteine protease can be found in a similar location with its side-chain hydrogen bonded to a backbone carbonyl oxygen in these three types of cysteine proteases. (B) The sequence motif around the nucleophilic Cys is highly conserved among alphaviral cysteine proteases. The side chain of Asn-475 is hydrogen bonded to Asp-507 and can be substituted by a Lys in other alphaviral nsP2 proteases, while in the SARS and MERS PLpro enzymes the Asn side chain is hydrogen bonded to the backbone carbonyl oxygen of a glycine.

In USP only one

hydrogen bond to the Asn is observed.

The location of the N-terminal residues near the first helix highlights another similarity between both papain and the nsP2pro (Suppl. Fig. S2). Comparison of the two structures shows better constraint of the residues N-terminal to the Cys in papain. One of papain's oxyanion hole residues (Gln-19) is within in this N-terminal segment (Figure 5). The VEEV nsP2 cysteine protease construct utilized for crystallization contains 20 residues N-terminal to Cys-477 while papain contains 24 residues. In the VEEV nsP2pro the Asn-475 side-chain appears to play an important role in stabilizing this N-terminal segment and preventing these residues from inhibiting the enzyme. In papain, a glycine is found at the same location (Gly23). Residues homologous to and functionally equivalent to Gln-19 have not been identified in the nsP2 proteases. The side-chains of Gln-471 and Asn-472 in the VEEV nsP2pro, while in a similar spatial location to Gln-19 in papain, are also rotated away from the active site in the alphaviral nsP2 protease structures that have been determined to date (PDB 2HWK,


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 33

5EZS, 5EZQ, 3TRK, 4GUA) (70-73). The more well-formed active site of papain and better stabilized transition state may account for the 500-fold difference in turnover numbers between papain (kcat = 2,520 min-1) and the VEEV nsP2pro (kcat = 5 min-1). From sequence alignments the Asn-475 of alphaviral nsP2 proteases is highly conserved and is substituted in only 2 of the alphaviral proteases by a Lys which may strengthen the interaction with Asp-507, a residue conserved in all alphaviral nsP2 proteases (Figures 2, 5). Viral proteases can have more than one function during infection; for instance the PLpro (papain-like proteases) of the coronaviruses, SARS (Severe Acute Respiratory Syndrome) and MERS (Middle East Respiratory Syndrome), can also function as K48 and K63 deubiquitinases (74). Tripartite motif proteins (TRIM), some of which are interferoninducible ubiquitin ligases (75), play an important role in the innate immune response and in exerting some of the interfering effects of IFN (76). While these substrates are seemingly disparate, in a Dali structural similarity search we found an Asn at the n-2 position (relative to the Cys) not only in the coronaviral cysteine proteases, but also in several papain-like USPs (Figure 5) (77). Based upon the Asn’s location in the active site, the side-chain of this residue was thought to directly stabilize the oxyanion and undergo a conformational change (rotate towards the oxyanion) in the SARS PLpro (Asn-110 (78)); however, in structures of the alphaviral nsP2 (PDB 5EZS (79)), coronaviral PLpro (PDB 4M0W (80)), and USPs (PDB 2IBI (81)), the side chain of the Asn is always found rotated away, even in the presence of substrate-mimics. In alphaviral cysteine proteases, Trp-478 was proposed to form part of the oxyanion hole (82), but appears to be too distant to stabilize the oxyanion in overlays of the N475A structure (PDB 6BCM) and the E64d-inhibited enzyme (PDB 5EZS).

22 ACS Paragon Plus Environment

Page 23 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

DISCUSSION In viral polyproteins several different enzymes are concatenated on a single polypeptide.

The multi-domained nature of nonstructural proteins presents significant

challenges in terms of protein expression, purification, and crystallization. While some domains can be expressed separately for inhibitor screening and structural analysis, the poorly constrained N-termini in the truncated constructs are often assumed to be unstructured linkers. However, these residues can affect the interpretation of mutagenesis experiments and the activity of the enzyme in unexpected ways. Here we describe a case where the tertiary structure of 20 N-terminal residues is affected by a single residue substitution, N475A. In other structures of the VEEV nsP2 cysteine protease (e.g. 2HWK, 5EZS, 5EZQ), the first 11 residues of the 20 are disordered.

The N475A substitution was expected to remove a

hydrogen bond donor and acceptor and allow us to determine if the side-chain was involved in substrate binding or oxyanion stabilization, a role proposed by others in the SARS PLpro (83). Unexpectedly, the mutation led to a self-inhibited state where the N-terminal residues were found to mimic the substrate. The h-bond made by the Asn-475 side chain to the Asp-507 backbone carbonyl appears to be critical in determining the orientation of the N-terminal 20 amino acids. While a flexible N-terminus might be expected to act as a competitive inhibitor, the N475A mutation had only minor effects on the Km (3.6-fold increase at pH 6.0 in 100 mM phosphate buffer and 1.3-fold increase in 50 mM HEPES pH 7.0 buffer). The case presented here is rather atypical in that the “competitive inhibitor” is comprised of residues which also form the active site (i.e. Cys-477) and substrate binding site. Analysis of the melting temperatures using CD spectroscopy showed a minor increase of 2 °C in the Tm of the N475A variant, but did not reveal the mode of inhibition; only the crystal structure was able to clarify the mechanism. The stabilization of the N-terminus is important since residues N-terminal of the 23 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 33

nucleophilic cysteine in papain and papain-like proteases are involved in TS-stabilization (Figure 5) and a precise organization of these residues is required for catalysis. Another example in the literature where a single mutation of a structurally important N-terminal residue affected the activity of a protease is that of the SARS 3CLpro N28A variant (84); this single mutation affected dimerization, the structure of active site residues, resulted in a significant loss of activity, but had no significant effect on the Km. The case here has some similarities to cysteine protease zymogens such as lysosomal proteases that block their active sites (85) and to toxin proteases that block their active sites with noncleavable pseudo substrates (86). There are two types of zymogens: those where the active site residues are in their active conformations before and after cleavage, and those where active site residues must rearrange shortly after cleavage to form a catalytically competent state (87). In this case the inhibitory pseudo substrate blocking peptide includes the nucleophilic cysteine, and rearrangement would be required. Additionally, it should be noted that the N-termini of both the A’ and B’ states do not overlay with the WT enzyme structures (PDB ID 2HWK, 5EZQ), a catalytically competent enzyme. They also do not overlay with the N-terminal residues of the E64d-inhibited enzyme (PDB ID 5EZS). The observed A’ and B’ states may both be inactive, but for different reasons. The C-terminal fusion of an inhibitory peptide to a protease has been used as a method of optimizing inhibitory peptide binding to a serine protease (88). Sorensen, et al. created a library of Back-Flip peptide-protease fusions containing 14-residue linkers with TEV-protease cleavage sites that could be cut to release the associated peptide inhibitor. In their work an increase in the Km was observed when the inhibitory peptide (nanomolar Ki) was fused to the enzyme. Thus, we initially expected to observe competitive inhibition and an increase in the Km for the N475A variant. While other explanations cannot be excluded, the minimal effects on Km may be consistent with several explanations.

Here, the re-


Page 25 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

organization of the N-terminal residues in the active site may account for the different effects on Km. If re-structuring of the active site of the N475A variant is not possible in the substrate-bound state or is very slow, the addition of excess substrate would not reverse the effects of inhibition. Substrate-induced conformational changes can also occur and can depend upon pH, temperature, and viscosity; these conditions differed between the structural vs. kinetic analyses. The pKa’s of active site residues are also dependent upon the proximity of adjacent residues and access to solvent. Substrate binding to the A’ state could lead to an ES complex where the conformation of the enzyme poorly catalyses the reaction due to poorly constrained active site and subsite residues during nucleophilic attack.

Another

possibility is that A’ and B’ states are not in rapid equilibrium with a state that is active (e.g. PDB ID 2HWK, 5EZQ); based on the structures one could consider this a case of mis-folding or hysteresis (89). The minimal change in Km may indicate that we are titrating a small fraction of the enzyme that is correctly folded and has detectable activity. In the multifunctional alphaviral nsP2, it is unclear as to whether any alternate conformations control the activity of the protease. Yost and Marcotrigiano reported that their attempts to model and dock the nsP2 protease onto the structure of the unprocessed nsP2/nsP3 complex (PDB 4GUA) resulted in steric clashes (90) suggesting that conformational changes must occur during cleavage in trans. In cells, the accessibility of the nsP2 cysteine protease’s active site has not been examined in the full length nsP2 protein or in the polyprotein. Thus, it is unclear as to whether the active site is blocked or accessible in these multi-domained globular structures, and if any of these domains undergo conformational changes to facilitate cleavage. Our structural similarity search showed that the Asn in the n-2 position relative to the nucleophilic Cys is conserved in three main types of cysteine proteases: (1) Clan CN alphaviral nsP2 cysteine proteases, (2) Clan CA coronaviral papain-like cysteine 25 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 33

proteases/deubquitinating enzymes, and (3) Clan CA papain-like human ubiquitin specific proteases. Mutation of Asn-475 to alanine leads to a significant decrease in kcat/Km; however, the role of the side-chain of this residue appears to be in stabilization of the fold, rather than stabilization of the oxyanion through direct hydrogen bonding.

ACKNOWLEDGMENTS. This work was funded by the Defense Threat Reduction Agency (DTRA) project numbers CB-SEED-SEED09-2-0061 and CBCall4-CBM-05-2-0019. The opinions expressed here are those of the authors and do not represent those of the U. S. Navy, U.S. Army, U. S. Department of Defense, or the U. S. government.

SUPPORTING INFORMATION PARAGRAPH. Supporting information includes a proposed mechanism, a structural comparison of papain and the VEEV nsP2 protease, and a table containing the melting temperatures of the WT and variant enzymes.


Page 27 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

REFERENCES. 1. Jose, J., Snyder, J. E., and Kuhn, R. J. (2009) A structural and functional perspective of alphavirus replication and assembly, Future. Microbiol. 4, 837-856. 2. Vasiljeva, L., Merits, A., Golubtsov, A., Sizemskaja, V., Kaariainen, L., and Ahola, T. (2003) Regulation of the sequential processing of Semliki Forest virus replicase polyprotein, J Biol. Chem. 278, 41636-41645. 3. Vasiljeva, L., Merits, A., Golubtsov, A., Sizemskaja, V., Kaariainen, L., and Ahola, T. (2003) Regulation of the sequential processing of Semliki Forest virus replicase polyprotein, J Biol. Chem. 278, 41636-41645. 4. Shirako, Y. and Strauss, J. H. (1994) Regulation of Sindbis virus RNA replication: uncleaved P123 and nsP4 function in minus-strand RNA synthesis, whereas cleaved products from P123 are required for efficient plus-strand RNA synthesis, J Virol. 68, 1874-1885. 5. Vasiljeva, L., Merits, A., Golubtsov, A., Sizemskaja, V., Kaariainen, L., and Ahola, T. (2003) Regulation of the sequential processing of Semliki Forest virus replicase polyprotein, J Biol. Chem. 278, 41636-41645. 6. Shin, G., Yost, S. A., Miller, M. T., Elrod, E. J., Grakoui, A., and Marcotrigiano, J. (2012) Structural and functional insights into alphavirus polyprotein processing and pathogenesis, Proc. Natl. Acad. Sci. U. S. A 109, 16534-16539. 7. Shin, G., Yost, S. A., Miller, M. T., Elrod, E. J., Grakoui, A., and Marcotrigiano, J. (2012) Structural and functional insights into alphavirus polyprotein processing and pathogenesis, Proc. Natl. Acad. Sci. U. S. A 109, 16534-16539. 8. Montgomery, S. A. and Johnston, R. E. (2007) Nuclear import and export of Venezuelan equine encephalitis virus nonstructural protein 2, J Virol. 81, 10268-10279. 9. Strauss, E. G., De Groot, R. J., Levinson, R., and Strauss, J. H. (1992) Identification of the active site residues in the nsP2 proteinase of Sindbis virus, Virology 191, 932-940. 10. Ding, M. X. and Schlesinger, M. J. (1989) Evidence that Sindbis virus NSP2 is an autoprotease which processes the virus nonstructural polyprotein, Virology 171, 280-284. 11. Hardy, W. R. and Strauss, J. H. (1989) Processing the nonstructural polyproteins of sindbis virus: nonstructural proteinase is in the C-terminal half of nsP2 and functions both in cis and in trans, J Virol. 63, 4653-4664. 12. Reichert, E., Clase, A., Bacetty, A., and Larsen, J. (2009) Alphavirus antiviral drug development: scientific gap analysis and prospective research areas, Biosecur. Bioterror. 7, 413427. 13. Ding, M. X. and Schlesinger, M. J. (1989) Evidence that Sindbis virus NSP2 is an autoprotease which processes the virus nonstructural polyprotein, Virology 171, 280-284. 14. Hardy, W. R. and Strauss, J. H. (1989) Processing the nonstructural polyproteins of sindbis virus: nonstructural proteinase is in the C-terminal half of nsP2 and functions both in cis and in trans, J Virol. 63, 4653-4664. 15. Strauss, E. G., De Groot, R. J., Levinson, R., and Strauss, J. H. (1992) Identification of the active site residues in the nsP2 proteinase of Sindbis virus, Virology 191, 932-940. 16. Merits, A., Vasiljeva, L., Ahola, T., Kaariainen, L., and Auvinen, P. (2001) Proteolytic processing of Semliki Forest virus-specific non-structural polyprotein by nsP2 protease, J Gen. Virol. 82, 765-773. 17. Rausalu, K., Utt, A., Quirin, T., Varghese, F. S., Zusinaite, E., Das, P. K., Ahola, T., and Merits, A. (2016) Chikungunya virus infectivity, RNA replication and non-structural polyprotein processing depend on the nsP2 proteaseâ€™s active site cysteine residue, Sci Rep 6, 37124. 18. Hu, X., Compton, J. R., Leary, D. H., Olson, M. A., Lee, M. S., Cheung, J., Ye, W., Ferrer, M., Southall, N., Jadhav, A., Morazzani, E. M., Glass, P. J., Marugan, J., and Legler, P. M. (2016)


27

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

19.

20.

21.

22.

23. 24.

25.

26. 27.

28.

29.

30.

31.

32.

33.

Page 28 of 33

Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease, Biochemistry 55, 3007-3019. Hu, X., Compton, J. R., Leary, D. H., Olson, M. A., Lee, M. S., Cheung, J., Ye, W., Ferrer, M., Southall, N., Jadhav, A., Morazzani, E. M., Glass, P. J., Marugan, J., and Legler, P. M. (2016) Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease, Biochemistry 55, 3007-3019. Hu, X., Compton, J. R., Leary, D. H., Olson, M. A., Lee, M. S., Cheung, J., Ye, W., Ferrer, M., Southall, N., Jadhav, A., Morazzani, E. M., Glass, P. J., Marugan, J., and Legler, P. M. (2016) Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease, Biochemistry 55, 3007-3019. Zhang, D., Tozser, J., and Waugh, D. S. (2009) Molecular cloning, overproduction, purification and biochemical characterization of the p39 nsp2 protease domains encoded by three alphaviruses, Protein Expr. Purif. 64, 89-97. Rausalu, K., Utt, A., Quirin, T., Varghese, F. S., Zusinaite, E., Das, P. K., Ahola, T., and Merits, A. (2016) Chikungunya virus infectivity, RNA replication and non-structural polyprotein processing depend on the nsP2 proteaseâ€™s active site cysteine residue, Sci Rep 6, 37124. Ding, M. X. and Schlesinger, M. J. (1989) Evidence that Sindbis virus NSP2 is an autoprotease which processes the virus nonstructural polyprotein, Virology 171, 280-284. Hardy, W. R. and Strauss, J. H. (1989) Processing the nonstructural polyproteins of sindbis virus: nonstructural proteinase is in the C-terminal half of nsP2 and functions both in cis and in trans, J Virol. 63, 4653-4664. Merits, A., Vasiljeva, L., Ahola, T., Kaariainen, L., and Auvinen, P. (2001) Proteolytic processing of Semliki Forest virus-specific non-structural polyprotein by nsP2 protease, J Gen. Virol. 82, 765-773. Russo, A. T., White, M. A., and Watowich, S. J. (2006) The crystal structure of the Venezuelan equine encephalitis alphavirus nsP2 protease, Structure. 14, 1449-1458. Hu, X., Compton, J. R., Leary, D. H., Olson, M. A., Lee, M. S., Cheung, J., Ye, W., Ferrer, M., Southall, N., Jadhav, A., Morazzani, E. M., Glass, P. J., Marugan, J., and Legler, P. M. (2016) Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease, Biochemistry 55, 3007-3019. Hu, X., Compton, J. R., Leary, D. H., Olson, M. A., Lee, M. S., Cheung, J., Ye, W., Ferrer, M., Southall, N., Jadhav, A., Morazzani, E. M., Glass, P. J., Marugan, J., and Legler, P. M. (2016) Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease, Biochemistry 55, 3007-3019. Hu, X., Compton, J. R., Leary, D. H., Olson, M. A., Lee, M. S., Cheung, J., Ye, W., Ferrer, M., Southall, N., Jadhav, A., Morazzani, E. M., Glass, P. J., Marugan, J., and Legler, P. M. (2016) Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease, Biochemistry 55, 3007-3019. Menard, R., Carriere, J., Laflamme, P., Plouffe, C., Khouri, H. E., Vernet, T., Tessier, D. C., Thomas, D. Y., and Storer, A. C. (1991) Contribution of the glutamine 19 side chain to transition-state stabilization in the oxyanion hole of papain, Biochemistry 30, 8924-8928. Zhang, W., Sulea, T., Tao, L., Cui, Q., Purisima, E. O., Vongsamphanh, R., Lachance, P., Lytvyn, V., Qi, H., Li, Y., and Menard, R. (2011) Contribution of active site residues to substrate hydrolysis by USP2: insights into catalysis by ubiquitin specific proteases, Biochemistry 50, 4775-4785. Hu, X., Compton, J. R., Leary, D. H., Olson, M. A., Lee, M. S., Cheung, J., Ye, W., Ferrer, M., Southall, N., Jadhav, A., Morazzani, E. M., Glass, P. J., Marugan, J., and Legler, P. M. (2016) Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease, Biochemistry 55, 3007-3019. Hu, X., Compton, J. R., Leary, D. H., Olson, M. A., Lee, M. S., Cheung, J., Ye, W., Ferrer, M., Southall, N., Jadhav, A., Morazzani, E. M., Glass, P. J., Marugan, J., and Legler, P. M. (2016) ACS Paragon Plus Environment

28

Page 29 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

34.

35.

36. 37. 38.

39.

40. 41.

42. 43. 44.

45. 46. 47.

48.

49.

Biochemistry

Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease, Biochemistry 55, 3007-3019. Hu, X., Compton, J. R., Leary, D. H., Olson, M. A., Lee, M. S., Cheung, J., Ye, W., Ferrer, M., Southall, N., Jadhav, A., Morazzani, E. M., Glass, P. J., Marugan, J., and Legler, P. M. (2016) Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease, Biochemistry 55, 3007-3019. Ruge, D. R., Dunning, F. M., Piazza, T. M., Molles, B. E., Adler, M., Zeytin, F. N., and Tucker, W. C. (2011) Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters, Anal. Biochem. 411, 200-209. Russo, A. T., White, M. A., and Watowich, S. J. (2006) The crystal structure of the Venezuelan equine encephalitis alphavirus nsP2 protease, Structure. 14, 1449-1458. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., and Read, R. J. (2007) Phaser crystallographic software, J Appl. Crystallogr. 40, 658-674. Murshudov, G. N., Skubak, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F., and Vagin, A. A. (2011) REFMAC5 for the refinement of macromolecular crystal structures, Acta Crystallogr. D. Biol Crystallogr. 67, 355-367. Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution, Acta Crystallogr. D. Biol Crystallogr. 66, 213-221. Emsley, P. and Cowtan, K. (2004) Coot: model-building tools for molecular graphics, Acta Crystallogr. D. Biol. Crystallogr. 60, 2126-2132. Hu, X., Compton, J. R., Leary, D. H., Olson, M. A., Lee, M. S., Cheung, J., Ye, W., Ferrer, M., Southall, N., Jadhav, A., Morazzani, E. M., Glass, P. J., Marugan, J., and Legler, P. M. (2016) Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease, Biochemistry 55, 3007-3019. Schechter, I. and Berger, A. (1967) On the size of the active site in proteases. I. Papain., Biochem. Biophys. Res. Commun. 27, 157-162. Russo, A. T., Malmstrom, R. D., White, M. A., and Watowich, S. J. (2010) Structural basis for substrate specificity of alphavirus nsP2 proteases, J Mol. Graph. Model. 29, 46-53. Hu, X., Compton, J. R., Leary, D. H., Olson, M. A., Lee, M. S., Cheung, J., Ye, W., Ferrer, M., Southall, N., Jadhav, A., Morazzani, E. M., Glass, P. J., Marugan, J., and Legler, P. M. (2016) Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease, Biochemistry 55, 3007-3019. Russo, A. T., White, M. A., and Watowich, S. J. (2006) The crystal structure of the Venezuelan equine encephalitis alphavirus nsP2 protease, Structure. 14, 1449-1458. Russo, A. T., Malmstrom, R. D., White, M. A., and Watowich, S. J. (2010) Structural basis for substrate specificity of alphavirus nsP2 proteases, J Mol. Graph. Model. 29, 46-53. Hu, X., Compton, J. R., Leary, D. H., Olson, M. A., Lee, M. S., Cheung, J., Ye, W., Ferrer, M., Southall, N., Jadhav, A., Morazzani, E. M., Glass, P. J., Marugan, J., and Legler, P. M. (2016) Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease, Biochemistry 55, 3007-3019. Hu, X., Compton, J. R., Leary, D. H., Olson, M. A., Lee, M. S., Cheung, J., Ye, W., Ferrer, M., Southall, N., Jadhav, A., Morazzani, E. M., Glass, P. J., Marugan, J., and Legler, P. M. (2016) Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease, Biochemistry 55, 3007-3019. Hu, X., Compton, J. R., Leary, D. H., Olson, M. A., Lee, M. S., Cheung, J., Ye, W., Ferrer, M., Southall, N., Jadhav, A., Morazzani, E. M., Glass, P. J., Marugan, J., and Legler, P. M. (2016) Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease, Biochemistry 55, 3007-3019. ACS Paragon Plus Environment

29

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 33

50. Hu, X., Compton, J. R., Leary, D. H., Olson, M. A., Lee, M. S., Cheung, J., Ye, W., Ferrer, M., Southall, N., Jadhav, A., Morazzani, E. M., Glass, P. J., Marugan, J., and Legler, P. M. (2016) Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease, Biochemistry 55, 3007-3019. 51. Russo, A. T., Malmstrom, R. D., White, M. A., and Watowich, S. J. (2010) Structural basis for substrate specificity of alphavirus nsP2 proteases, J Mol. Graph. Model. 29, 46-53. 52. Russo, A. T., White, M. A., and Watowich, S. J. (2006) The crystal structure of the Venezuelan equine encephalitis alphavirus nsP2 protease, Structure. 14, 1449-1458. 53. Russo, A. T., Malmstrom, R. D., White, M. A., and Watowich, S. J. (2010) Structural basis for substrate specificity of alphavirus nsP2 proteases, J Mol. Graph. Model. 29, 46-53. 54. Lecaille, F., Authie, E., Moreau, T., Serveau, C., Gauthier, F., and Lalmanach, G. (2001) Subsite specificity of trypanosomal cathepsin L-like cysteine proteases. Probing the S2 pocket with phenylalanine-derived amino acids, Eur. J Biochem 268, 2733-2741. 55. Hedstrom, L. (2002) Serine protease mechanism and specificity, Chem Rev 102, 4501-4524. 56. Hu, X., Compton, J. R., Leary, D. H., Olson, M. A., Lee, M. S., Cheung, J., Ye, W., Ferrer, M., Southall, N., Jadhav, A., Morazzani, E. M., Glass, P. J., Marugan, J., and Legler, P. M. (2016) Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease, Biochemistry 55, 3007-3019. 57. Hook, V. Y., Kindy, M., and Hook, G. (2008) Inhibitors of cathepsin B improve memory and reduce beta-amyloid in transgenic Alzheimer disease mice expressing the wild-type, but not the Swedish mutant, beta-secretase site of the amyloid precursor protein, J Biol Chem 283, 77457753. 58. Hu, X., Compton, J. R., Leary, D. H., Olson, M. A., Lee, M. S., Cheung, J., Ye, W., Ferrer, M., Southall, N., Jadhav, A., Morazzani, E. M., Glass, P. J., Marugan, J., and Legler, P. M. (2016) Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease, Biochemistry 55, 3007-3019. 59. Zhang, D., Tozser, J., and Waugh, D. S. (2009) Molecular cloning, overproduction, purification and biochemical characterization of the p39 nsp2 protease domains encoded by three alphaviruses, Protein Expr. Purif. 64, 89-97. 60. Hu, X., Compton, J. R., Leary, D. H., Olson, M. A., Lee, M. S., Cheung, J., Ye, W., Ferrer, M., Southall, N., Jadhav, A., Morazzani, E. M., Glass, P. J., Marugan, J., and Legler, P. M. (2016) Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease, Biochemistry 55, 3007-3019. 61. Hu, X., Compton, J. R., Leary, D. H., Olson, M. A., Lee, M. S., Cheung, J., Ye, W., Ferrer, M., Southall, N., Jadhav, A., Morazzani, E. M., Glass, P. J., Marugan, J., and Legler, P. M. (2016) Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease, Biochemistry 55, 3007-3019. 62. Hu, X., Compton, J. R., Leary, D. H., Olson, M. A., Lee, M. S., Cheung, J., Ye, W., Ferrer, M., Southall, N., Jadhav, A., Morazzani, E. M., Glass, P. J., Marugan, J., and Legler, P. M. (2016) Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease, Biochemistry 55, 3007-3019. 63. Rausalu, K., Utt, A., Quirin, T., Varghese, F. S., Zusinaite, E., Das, P. K., Ahola, T., and Merits, A. (2016) Chikungunya virus infectivity, RNA replication and non-structural polyprotein processing depend on the nsP2 proteaseâ€™s active site cysteine residue, Sci Rep 6, 37124. 64. Hu, X., Compton, J. R., Leary, D. H., Olson, M. A., Lee, M. S., Cheung, J., Ye, W., Ferrer, M., Southall, N., Jadhav, A., Morazzani, E. M., Glass, P. J., Marugan, J., and Legler, P. M. (2016) Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease, Biochemistry 55, 3007-3019. 65. Rees, D. C. and Robertson, A. D. (2001) Some thermodynamic implications for the thermostability of proteins, Protein Sci 10, 1187-1194. ACS Paragon Plus Environment

30

Page 31 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

66. Golubtsov, A., Kaariainen, L., and Caldentey, J. (2006) Characterization of the cysteine protease domain of Semliki Forest virus replicase protein nsP2 by in vitro mutagenesis, FEBS Lett 580, 1502-1508. 67. Barrett, A. J. and Rawlings, N. D. Evolutionary lines of cysteine peptidases. 68. Russo, A. T., White, M. A., and Watowich, S. J. (2006) The crystal structure of the Venezuelan equine encephalitis alphavirus nsP2 protease, Structure. 14, 1449-1458. 69. Russo, A. T., White, M. A., and Watowich, S. J. (2006) The crystal structure of the Venezuelan equine encephalitis alphavirus nsP2 protease, Structure. 14, 1449-1458. 70. Russo, A. T., White, M. A., and Watowich, S. J. (2006) The crystal structure of the Venezuelan equine encephalitis alphavirus nsP2 protease, Structure. 14, 1449-1458. 71. Hu, X., Compton, J. R., Leary, D. H., Olson, M. A., Lee, M. S., Cheung, J., Ye, W., Ferrer, M., Southall, N., Jadhav, A., Morazzani, E. M., Glass, P. J., Marugan, J., and Legler, P. M. (2016) Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease, Biochemistry 55, 3007-3019. 72. Cheung, J., Franklin, M., Mancia, F., Rudolph, M., Cassidy, M., Gary, E., Burshteyn, F., and Love, J. (2011) Structure of the Chikungunya virus nsP2 protease. 73. Shin, G., Yost, S. A., Miller, M. T., Elrod, E. J., Grakoui, A., and Marcotrigiano, J. (2012) Structural and functional insights into alphavirus polyprotein processing and pathogenesis, Proc. Natl. Acad. Sci. U. S. A 109, 16534-16539. 74. Yang, X., Chen, X., Bian, G., Tu, J., Xing, Y., Wang, Y., and Chen, Z. (2014) Proteolytic processing, deubiquitinase and interferon antagonist activities of Middle East respiratory syndrome coronavirus papain-like protease, J Gen. Virol. 95, 614-626. 75. Carthagena, L., Bergamaschi, A., Luna, J. M., David, A., Uchil, P. D., Margottin-Goguet, F., Mothes, W., Hazan, U., Transy, C., Pancino, G., and Nisole, S. Ã. (2009) Human TRIM Gene Expression in Response to Interferons, PLoS One 4, e4894. 76. Kawai, T. and Akira, S. (2011) Regulation of innate immune signalling pathways by the tripartite motif (TRIM) family proteins, EMBO Mol Med 3, 513-527. 77. Zhang, W., Sulea, T., Tao, L., Cui, Q., Purisima, E. O., Vongsamphanh, R., Lachance, P., Lytvyn, V., Qi, H., Li, Y., and Menard, R. (2011) Contribution of active site residues to substrate hydrolysis by USP2: insights into catalysis by ubiquitin specific proteases, Biochemistry 50, 4775-4785. 78. Ratia, K., Saikatendu, K. S., Santarsiero, B. D., Barretto, N., Baker, S. C., Stevens, R. C., and Mesecar, A. D. (2006) Severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme, Proc. Natl. Acad. Sci. U. S. A 103, 5717-5722. 79. Hu, X., Compton, J. R., Leary, D. H., Olson, M. A., Lee, M. S., Cheung, J., Ye, W., Ferrer, M., Southall, N., Jadhav, A., Morazzani, E. M., Glass, P. J., Marugan, J., and Legler, P. M. (2016) Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease, Biochemistry 55, 3007-3019. 80. Chou, C. Y., Lai, H. Y., Chen, H. Y., Cheng, S. C., Cheng, K. W., and Chou, Y. W. (2014) Structural basis for catalysis and ubiquitin recognition by the severe acute respiratory syndrome coronavirus papain-like protease, Acta Crystallogr. D. Biol. Crystallogr. 70, 572-581. 81. Walker, J. R., Avvakumov, G. V., Bernstein, G., Xue, S., Finerty Jr., P. J., MacKenzie, F., Weigelt, J., Sundstrom, M., Arrowsmith, C. H., Edwards, A. M., Bochkarev, A., and DhePaganon, S. (6 A.D.) Covalent Ubiquitin-USP2 Complex, PDB 2IBI ed.. 82. Russo, A. T., Malmstrom, R. D., White, M. A., and Watowich, S. J. (2010) Structural basis for substrate specificity of alphavirus nsP2 proteases, J Mol. Graph. Model. 29, 46-53. 83. Ratia, K., Saikatendu, K. S., Santarsiero, B. D., Barretto, N., Baker, S. C., Stevens, R. C., and Mesecar, A. D. (2006) Severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme, Proc. Natl. Acad. Sci. U. S. A 103, 5717-5722.


31

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 33

84. Barrila, J., Gabelli, S. B., Bacha, U., Amzel, L. M., and Freire, E. (2010) Mutation of Asn28 disrupts the dimerization and enzymatic activity of SARS 3CL(pro), Biochemistry 49, 43084317. 85. Turk, V., Stoka, V., Vasiljeva, O., Renko, M., Sun, T., Turk, B., and Turk, D. (2012) Cysteine cathepsins: from structure, function and regulation to new frontiers, Biochim. Biophys Acta 1824, 68-88. 86. Chen, S., Kim, J. J., and Barbieri, J. T. (2007) Mechanism of substrate recognition by botulinum neurotoxin serotype A, J Biol Chem 282, 9621-9627. 87. Renatus, M., Stennicke, H. R., Scott, F. L., Liddington, R. C., and Salvesen, G. S. (2001) Dimer formation drives the activation of the cell death protease caspase 9, Proc Natl Acad Sci U S A 98, 14250-14255. 88. Sorensen, H. P., Xu, P., Jiang, L., Kromann-Hansen, T., Jensen, K. J., Huang, M., and Andreasen, P. A. (2015) Selection of High-Affinity Peptidic Serine Protease Inhibitors with Increased Binding Entropy from a Back-Flip Library of Peptide-Protease Fusions, J Mol Biol 427, 3110-3122. 89. Hanozet, G. F., Pircher HP, F. A. U., Vanni, P. F., Oesch, B. F., and Semenza, G. An example of enzyme hysteresis. The slow and tight interaction of some fully competitive inhibitors with small intestinal sucrase. 90. Yost, S. A. and Marcotrigiano, J. (2013) Viral precursor polyproteins: keys of regulation from replication to maturation, Curr. Opin. Virol 3, 137-142.


32

Page 33 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

SYNOPSIS TOC. The nsP2 cysteine protease is a validated target for drug discovery. Mutation of a single conserved motif residue, Asn-475, of the alphaviral nsP2 cysteine protease from the Venezuelan Equine Encephalitis virus (VEEV) led to a large conformational change. The N-terminus rotated into the substrate binding cleft of the protease, mimicking the substrate. Interestingly, the N-terminus was not hydrolyzed, consistent with a catalytically unproductive state.


33

Mutation of Asn-475 in the Venezuelan Equine Encephalitis Virus

Recommend Documents