The Molecular Mechanism of Domain Swapping of the C-Terminal Domain of the SARS-Coronavirus Main Protease

Terse, Vishram L.; Gosavi, Shachi

doi:10.1016/j.bpj.2020.11.2277

Cited by 5 publications

(16 citation statements)

References 86 publications

(124 reference statements)

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“…The overall order of helix packing (α1 structuring early followed by α4 with α5 forming last) is similar to the order of helix stabilities seen in NMR spectroscopy studies of SARS-1 M pro C . Thus, the folding of SARS-2 M pro C seen in simulations is similar to that of SARS-1 M pro C as seen in both simulations and experiments. , Specifically, both SARS-1 M pro C and SARS-2 M pro C fold cooperatively, and there is an order to the α-helix formation potentially due to a difference in the helix stabilities. , It has previously been shown that a self-peptide containing the folding nucleus (residues that gain structure early during folding) of ubiquitin can modulate its folding by replacing ubiquitin’s own nucleus . Since SARS-2 M pro C has localized early structure formation (Figure E), akin to a polarized folding nucleus, , we tested if self-peptides of SARS-2 M pro C could insert into the whole protein during folding similar to ubiquitin.…”

Section: Resultssupporting

confidence: 67%

“…SARS-2 M pro C (Figure A,B) is structurally similar to SARS-1 M pro C with their sequences differing only at 4 residues (Figure S1). Although little residue specific information about the folding of SARS-2 M pro C is available, given their similarity, it is expected that SARS-2 M pro C will fold in a manner similar to SARS-1 M pro C. We test this by performing simulations of a Cα-SBM of SARS-2 M pro C (see Methods) based on a previous Cα-SBM of SARS-1 M pro C whose folding broadly agrees with SARS-1 M pro C folding experiments. , The native contact map of SARS-2 M pro C used in the Cα-SBM is shown in Figure C. The SARS-2 M pro C Cα-SBM simulations performed at the folding temperature, T normal f , were analyzed using the number of formed native contacts ( Q ) as a progress coordinate.…”

Section: Resultsmentioning

confidence: 99%

“…The number or fraction of formed native contacts in a given simulation snapshot is often used as an order parameter to study protein folding. , Since the number of formed native contacts, Q , has previously been used to study the folding of SARS-1 M pro C, we use it here to study the folding of the homologous SARS-2 M pro C. A native contact was considered to be formed in a simulation snapshot if the two residues in contact were within 1.2 times their distance in the native structure. The total number of formed native contacts ( Q ) was counted for every snapshot in the trajectory data.…”

Section: Methodsmentioning

confidence: 99%

“…This Cα-SBM has previously been used to study the folding and domainswapping of the structurally homologous helical SARS-1 M pro C. These studies showed, 34 in agreement with experiments, 35 that there was an order to the formation of the helices in SARS-1 M pro C. Further, early structure formation was concentrated in specific regions of the protein. 34 Our folding simulations of SARS-2 M pro C showed an order of helix formation similar to that in SARS-1 M pro C. Simulations of SARS-2 M pro C with self-peptides spanning the individual helices and a long loop between helix α4 and α5 (loop4) showed that only self-peptides with residues that participate in early forming contacts (α1 and loop4) are able to insert into the protein and replace the corresponding native segments (Figure 3C,E). We then discuss the potential for α1 and loop4 to be inhibitory peptides using previously observed structure− function relationships in M pro .…”

Section: ■ Introductionmentioning

confidence: 99%

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Potential Self-Peptide Inhibitors of the SARS-CoV-2 Main Protease

Banerjee

Gosavi

2023

J. Phys. Chem. B

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The SARS-CoV-2 main protease (M pro ) plays an essential role in viral replication, cleaving viral polyproteins into functional proteins. This makes M pro an important drug target. M pro consists of an N-terminal catalytic domain and a C-terminal α-helical domain (M pro C). Previous studies have shown that peptides derived from a given protein sequence (self-peptides) can affect the folding and, in turn, the function of that protein. Since the SARS-CoV-1 M pro C is known to stabilize its M pro and regulate its function, we hypothesized that SARS-CoV-2 M pro C-derived self-peptides may modulate the folding and the function of SARS-CoV-2 M pro . To test this, we studied the folding of M pro C in the presence of various selfpeptides using coarse-grained structure-based models and molecular dynamics simulations. In these simulations of M pro C and one selfpeptide, we found that two self-peptides, the α1-helix and the loop between α4 and α5 (loop4), could replace the equivalent native sequences in the M pro C structure. Replacement of either sequence in full-length M pro should, in principle, be able to perturb M pro function albeit through different mechanisms. Some general principles for the rational design of self-peptide inhibitors emerge: The simulations show that prefolded self-peptides are more likely to replace native sequences than those which do not possess structure. Additionally, the α1-helix self-peptide is kinetically stable and once inserted rarely exchanges with the native α1-helix, while the loop4 self-peptide is easily replaced by the native loop4, making it less useful for modulating function. In summary, a prefolded α1derived peptide should be able to inhibit SARS-CoV-2 M pro function.

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Section: Resultssupporting

confidence: 67%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

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Potential Self-Peptide Inhibitors of the SARS-CoV-2 Main Protease

Banerjee

Gosavi

2023

J. Phys. Chem. B

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“…The minimum for the harmonic term was set to the distance between the centers of mass of the two monomers in the folded dimer (WT CP2: 0.16 nm, CiP CP2: 0.22 nm). Previous simulations have used this technique to prevent unfolded monomers from drifting too far from each other. − Although this is a weak harmonic constraint, the values of the force constant and the centers of mass distance do marginally affect the height of the free energy barrier with an increase in barrier height seen at lower force constants. However, the change in parameter values does not affect the folding mechanism (see the Supporting Information Figures S3–S5).…”

Section: Methodsmentioning

confidence: 99%

Understanding the Folding Mediated Assembly of the Bacteriophage MS2 Coat Protein Dimers

Prakash

Gosavi

2021

J. Phys. Chem. B

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The capsids of RNA viruses such as MS2 are great models for studying protein self-assembly because they are made almost entirely of multiple copies of a single coat protein (CP). Although CP is the minimal repeating unit of the capsid, previous studies have shown that CP exists as a homodimer (CP2) even in an acid-disassembled system, indicating that CP2 is an obligate dimer. Here, we investigate the molecular basis of this obligate dimerization using coarse-grained structure-based models and molecular dynamics simulations. We find that, unlike monomeric proteins of similar size, CP populates a single partially folded ensemble whose “foldedness” is sensitive to denaturing conditions. In contrast, CP2 folds similarly to single-domain proteins populating only the folded and the unfolded ensembles, separated by a prominent folding free energy barrier. Several intramonomer contacts form early, but the CP2 folding barrier is crossed only when the intermonomer contacts are made. A dissection of the structure of CP2 through mutant folding simulations shows that the folding barrier arises both from the topology of CP and the interface contacts of CP2. Together, our results show that CP2 is an obligate dimer because of kinetic stability, that is, dimerization induces a folding barrier and that makes it difficult for proteins in the dimer minimum to partially unfold and access the monomeric state without completely unfolding. We discuss the advantages of this obligate dimerization in the context of dimer design and virus stability.

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Tandem repeats of highly bioluminescent NanoLuc are refolded noncanonically by the Hsp70 machinery

Apostolidou,

Zhang,

Pandya

et al. 2024

Protein Science

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Chaperones are a large family of proteins crucial for maintaining cellular protein homeostasis. One such chaperone is the 70 kDa heat shock protein (Hsp70), which plays a crucial role in protein (re)folding, stability, functionality, and translocation. While the key events in the Hsp70 chaperone cycle are well established, a relatively small number of distinct substrates were repetitively investigated. This is despite Hsp70 engaging with a plethora of cellular proteins of various structural properties and folding pathways. Here we analyzed novel Hsp70 substrates, based on tandem repeats of NanoLuc (Nluc), a small and highly bioluminescent protein with unique structural characteristics. In previous mechanical unfolding and refolding studies, we have identified interesting misfolding propensities of these Nluc‐based tandem repeats. In this study, we further investigate these properties through in vitro bulk experiments. Similar to monomeric Nluc, engineered Nluc dyads and triads proved to be highly bioluminescent. Using the bioluminescence signal as the proxy for their structural integrity, we determined that heat‐denatured Nluc dyads and triads can be efficiently refolded by the E. coli Hsp70 chaperone system, which comprises DnaK, DnaJ, and GrpE. In contrast to previous studies with other substrates, we observed that Nluc repeats can be efficiently refolded by DnaK and DnaJ, even in the absence of GrpE co‐chaperone. Taken together, our study offers a new powerful substrate for chaperone research and raises intriguing questions about the Hsp70 mechanisms, particularly in the context of structurally diverse proteins.

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The Molecular Mechanism of Domain Swapping of the C-Terminal Domain of the SARS-Coronavirus Main Protease

Cited by 5 publications

References 86 publications

Potential Self-Peptide Inhibitors of the SARS-CoV-2 Main Protease

Potential Self-Peptide Inhibitors of the SARS-CoV-2 Main Protease

Understanding the Folding Mediated Assembly of the Bacteriophage MS2 Coat Protein Dimers

Tandem repeats of highly bioluminescent NanoLuc are refolded noncanonically by the Hsp70 machinery

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