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001-es BibID:	BIBFORM120789
035-os BibID:	(Scopus)85191248548 (WoS)001208395200001
Első szerző:	Golda Mária (molekuláris biológus)
Cím:	P1' specificity of the S219V/R203G mutant tobacco etch virus protease / Mária Golda, Gyula Hoffka, Scott Cherry, Joseph E. Tropea, George T. Lountos, David S. Waugh, Alexander Wlodawer, József Tőzsér, János András Mótyán
Dátum:	2024
ISSN:	0887-3585
Megjegyzések:	Proteases that recognize linear amino acid sequences with high specificity became indispensable tools of recombinant protein technology for the removal of various fusion tags. Due to its stringent sequence specificity, the catalytic domain of the nuclear inclusion cysteine protease of tobacco etch virus (TEV PR) is also a widely applied reagent for enzymatic removal of fusion tags. For this reason, efforts have been made to improve its stability and modify its specificity. For example, P1' autoproteolytic cleavage-resistant mutant (S219V) TEV PR was found not only to be nearly impervious to self-inactivation, but also exhibited greater stability and catalytic efficiency than the wild-type enzyme. An R203G substitution has been reported to further relax the P1' specificity of the enzyme, however, these results were obtained from crude intracellular assays. Until now, there has been no rigorous comparison of the P1' specificity of the S219V and S219V/R203G mutants in vitro, under carefully controlled conditions. Here, we compare the P1' amino acid preferences of these single and double TEV PR mutants. The in vitro analysis was performed by using recombinant protein substrates representing 20 P1' variants of the consensus TENLYFQ*SGT cleavage site, and synthetic oligopeptide substrates were also applied to study a limited set of the most preferred variants. In addition, the enzyme-substrate interactions were analyzed in silico. The results indicate highly similar P1' preferences for both enzymes, many side-chains can be accommodated by the S1' binding sites, but the kinetic assays revealed lower catalytic efficiency for the S219V/R203G than for the S219V mutant.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk tobacco etch virus protein structure molecular dynamics fusion tag removal enzymology TEV protease protease Proteolysis
Megjelenés:	Proteins-Structure Function And Bioinformatics. - [Epub ahead of print] (2024). -
További szerzők:	Hoffka Gyula (1992-) (vegyész) Cherry, Scott Tropea, Joseph E. Lountos, George T. Waugh, David S. Wlodawer, Alexander Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész) Mótyán János András (1981-) (biokémikus, molekuláris biológus)
Pályázati támogatás:	TKP2021-EGA-20 Egyéb ÚNKP-23-5-DE-486 Egyéb BO/00110/23/5 MTA 75N91019D00024 Egyéb
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001-es BibID:	BIBFORM108790
035-os BibID:	(Scopus)85148381582 (WoS)000944102100001
Első szerző:	Hoffka Gyula (vegyész)
Cím:	Self-inhibited state of Venezuelan equine encephalitis virus (VEEV) nsP2 cysteine protease : a crystallographic and molecular dynamics analysis / Gyula Hoffka, George T. Lountos, Danielle Needle, Alexander Wlodawer, David S. Waugh, József Tőzsér, János András Mótyán
Dátum:	2023
ISSN:	0022-2836
Megjegyzések:	The Venezuelan equine encephalitis virus (VEEV) belongs to the Togaviridae family and is pathogenic to both humans and equines. The VEEV non-structural protein 2 (nsP2) is a cysteine protease (nsP2pro) that processes the polyprotein and thus it is a drug target for inhibitor discovery. The atomic structure of the VEEV nsP2 catalytic domain was previously characterized by both X-ray crystallography and computational studies. A modified nsP2pro harboring a N475A mutation in the N terminus was observed to exhibit an unexpected conformation: the N-terminal residues bind to the active site, mimicking binding of a substrate. The large conformational change of the N terminus was assumed to be induced by the N475A mutation, as N475 has an important role in stabilization of the N terminus and the active site. This conformation was first observed in the N475A mutant, but we also found it while determining a crystal structure of the catalytically active nsP2pro containing the wild-type N475 active site residue and K741A/K767A surface entropy reduction mutations. This suggests that the N475A mutation is not a prerequisite for self-inhibition. Here, we describe a high resolution (1.46 ?A) crystal structure of a truncated nsP2pro (residues 463-785, K741A/K767A) and analyze the structure further by molecular dynamics to study the active and self-inhibited conformations of nsP2pro and its N475A mutant. A comparison of the different conformations of the N-terminal residues sheds a light on the interactions that play an important role in the stabilization of the enzyme.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk Venezuelan equine encephalitis virus protease alphavirus crystallography molecular dynamics
Megjelenés:	Journal Of Molecular Biology. - 435 : 6 (2023), p. 1-20. -
További szerzők:	Lountos, George T. Needle, Danielle Wlodawer, Alexander Waugh, David S. Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész) Mótyán János András (1981-) (biokémikus, molekuláris biológus)
Pályázati támogatás:	TKP2021-EGA-20 (Biotechnology) Egyéb
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