Structural Analysis of Sindbis Virus Capsid Mutants Involving Assembly and Catalysis

Choi, Hojong; Lee, Sukyeong; Zhang, Yanping; McKinney, B. R.; Wengler, Gerd; Rossmann, Michael G.; Kühn, Richard

doi:10.1006/jmbi.1996.0505

Cited by 53 publications

(60 citation statements)

References 41 publications

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“…In the AVCP⌬2 structure, both of the monomers are in tail-to-tail contact through their C-terminal subdomains. Previously, a number of dimers and trimers were found in the SCP and the SFV CPs that were arranged in head-to-tail and tail-to-tail fashion in different crystal forms (2,13,19). In headto-tail dimers, the N-and C-terminal subdomains are in contact, as in the SCP S215A mutant crystal structure (13).…”

Section: Resultsmentioning

confidence: 98%

“…Previously, a number of dimers and trimers were found in the SCP and the SFV CPs that were arranged in head-to-tail and tail-to-tail fashion in different crystal forms (2,13,19). In headto-tail dimers, the N-and C-terminal subdomains are in contact, as in the SCP S215A mutant crystal structure (13). In head-tohead dimer formation, the N-terminal subdomains are in contact, as in the SFV CP wild-type I crystal type (19).…”

Section: Resultsmentioning

confidence: 99%

“…The active-site molecular architecture and the catalytic triad are well conserved among all serine proteases including alphavirus CPs (AVCP His144, Asp163, and Ser218) and exhibit similar spatial architectures in the active site (2,16,17). Additionally, the GDSG motif containing the active-site nucleophilic serine residue that is well conserved in chymotrypsin-like serine proteases is also found to be completely conserved in the CP of alphaviruses (AVCP 215 GDSG 219 ) (2,13,14,16).…”

mentioning

confidence: 99%

“…After cleavage, the free carboxylic group of conserved Trp267 at the P 1 position interacts with the catalytic triad and remains bound in the S 1 pocket of the CP. This bound carboxyl terminus tryptophan residue in the active site inactivates the protease and blocks further trans-cleavage activity (2,(13)(14)(15)(16). The active-site molecular architecture and the catalytic triad are well conserved among all serine proteases including alphavirus CPs (AVCP His144, Asp163, and Ser218) and exhibit similar spatial architectures in the active site (2,16,17).…”

mentioning

confidence: 99%

“…Additionally, the crystal structure of catalytically inactive S215A variant of Sindbis virus capsid protein (SCP) is also available, in which two additional C-terminal P 1 = and P 2 = residues (Ser265 and Ala266) are also bound near the active site (13). As the structure consists of substrate residues in the specificity pocket, this structure is considered an enzyme-substrate complex form of CP in which the catalytic triad residue Ser215 has been mutated to alanine to abolish the self-cleavage activity.…”

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confidence: 99%

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trans -Protease Activity and Structural Insights into the Active Form of the Alphavirus Capsid Protease

Aggarwal

Dhindwal

Kumar

et al. 2014

J Virol

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The alphavirus capsid protein (CP) is a serine protease that possesses cis-proteolytic activity essential for its release from the nascent structural polyprotein. The released CP further participates in viral genome encapsidation and nucleocapsid core formation, followed by its attachment to glycoproteins and virus budding. Thus, protease activity of the alphavirus capsid is a potential antialphaviral target to arrest capsid release, maturation, and structural polyprotein processing. However, the discovery of capsid protease inhibitors has been hampered due to the lack of a suitable screening assay and of the crystal structure in its active form. Here, we report the development of a trans-proteolytic activity assay for Aura virus capsid protease (AVCP) based on fluorescence resonance energy transfer (FRET) for screening protease inhibitors. Kinetic parameters using fluorogenic peptide substrates were estimated, and the K m value was found to be 2.63 ؎ 0.62 M while the k cat /K m value was 4.97 ؋ 10 4 M ؊1 min ؊1 . Also, the crystal structure of the trans-active form of AVCP has been determined to 1.81-Å resolution. Structural comparisons of the active form with the crystal structures of available substrate-bound mutant and inactive blocked forms of the capsid protease identify conformational changes in the active site, the oxyanion hole, and the substrate specificity pocket residues, which could be critical for rational drug design. IMPORTANCEThe alphavirus capsid protease is an attractive antiviral therapeutic target. In this study, we have described the formerly unap- , the disordered C-terminal residues after His261, and the presence of a water molecule in the oxyanion hole of AVCP⌬2 (AVCP with a deletion of the last two residues at the C terminus) reveal the effect of the C-terminal Trp267 deletion on enzyme structure. New structural data reported in this study along with the fluorogenic assay will be useful in substrate specificity characterization, high-throughput protease inhibitor screening, and structure-based development of antiviral drugs.

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Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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trans -Protease Activity and Structural Insights into the Active Form of the Alphavirus Capsid Protease

Aggarwal

Dhindwal

Kumar

et al. 2014

J Virol

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Structure of Semliki Forest virus core protein

Choi

Lee

et al. 1997

Proteins

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Alphaviruses are enveloped, insect-borne viruses, which contains a positive-sense RNA genome. The protein capsid is surrounded by a lipid membrane, which is penetrated by glycoprotein spikes. The structure of the Sindbis virus (SINV) (the type virus) core protein (SCP) was previously determined and found to have a chymotrypsin-like structure. SCP is a serine proteinase which cleaves itself from a polyprotein. Semliki Forest virus (SFV) is among the most distantly related alphaviruses to SINV. Similar to SCP, autocatalysis is inhibited in SFCP after cleavage of the polyprotein by leaving the carboxy-terminal tryptophan in the specificity pocket. The structures of two different crystal forms (I and II) of SFV core protein (SFCP) have been determined to 3.0 A and 3.3 A resolution, respectively. The SFCP monomer backbone structure is very similar to that of SCP. The dimeric association between monomers, A and B, found in two different crystal forms of SCP is also present in both crystal forms of SFCP. However, a third monomer, C, occurs in SFCP crystal form I. While monomers A and B make a tail-to-tail dimer contact, monomers B and C make a head-to-head dimer contact. A hydrophobic pocket on the surface of the capsid protein, the proposed site of binding of the E2 glycoprotein, has large conformational differences with respect to SCP and, in contrast to SCP, is found devoid of bound peptide. In particular, Tyr184 is pointing out of the hydrophobic pocket in SFCP, whereas the equivalent tyrosine in SCP is pointing into the pocket. The conformation of Tyr184, found in SFCP, is consistent with its availability for iodination, as observed in the homologous SINV cores. This suggests, by comparison with SCP, that E2 binding to cores causes major conformational changes, including the burial of Tyr184, which would stabilize the intact virus on budding from an infected cell. The head-to-tail contacts found in the pentameric and hexameric associations within the virion utilize in the same monomer surface regions as found in the crystalline dimer interfaces.

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Probing the potential glycoprotein binding site of sindbis virus capsid protein with dioxane and model building

1998

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Alphavirus budding from the plasma membrane is initiated by the specific interaction of the nucleocapsid with the cytoplasmic domain of the glycoprotein E2. It was proposed (Lee et al., Structure 4:531-541, 1996) that binding of the capsid protein residues 108 to 110 (the "N-terminal arm" residues) to a hydrophobic pocket on the surface of the neighboring capsid protein in the crystal structure mimics the binding of the E2 C-terminal residues into this pocket. In addition, structural comparisons of wild-type and mutant Sindbis virus capsid protein (SCP) and Semliki Forest virus capsid protein suggested that budding is associated with a switch between two conformations of the hydrophobic pocket. To test the proposed mechanism, SCP(114-264), which is missing the N-terminal arm, was crystallized to examine the pocket conformation when the pocket is empty. However, the pocket was occupied by dioxane molecules from the crystallization solution. The pocket conformation was the same as that when it was occupied by the N-terminal arm, demonstrating that the pocket favors binding ligands of appropriate size and shape.

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Structural Analysis of Sindbis Virus Capsid Mutants Involving Assembly and Catalysis

Cited by 53 publications

References 41 publications

trans -Protease Activity and Structural Insights into the Active Form of the Alphavirus Capsid Protease

trans -Protease Activity and Structural Insights into the Active Form of the Alphavirus Capsid Protease

Structure of Semliki Forest virus core protein

Probing the potential glycoprotein binding site of sindbis virus capsid protein with dioxane and model building

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