The Active Site of a Lon Protease fromMethanococcus jannaschiiDistinctly Differs from the Canonical Catalytic Dyad of Lon Proteases

Abstract Rhomboids are ubiquitous intramembrane serine proteases involved in various signaling pathways. While the high-resolution structures of the Escherichia coli rhomboid GlpG with various inhibitors revealed an active site comprised of a serine-histidine dyad and an extensive oxyanion hole, the molecular details of rhomboid catalysis were unclear because substrates are unknown for most of the family members. Here we used the only known physiological pair of AarA rhomboid with its psTatA substrate to decipher the contribution of catalytically important residues to the reaction rate enhancement. An MD-refined homology model of AarA was used to identify residues important for catalysis. We demonstrated that the AarA active site geometry is strict and intolerant to alterations. We probed the roles of H83 and N87 oxyanion hole residues and determined that substitution of H83 either abolished AarA activity or reduced the transition state stabilization energy (ΔΔG‡) by 3.1 kcal/mol; substitution of N87 decreased ΔΔG‡ by 1.6–3.9 kcal/mol. Substitution M154, a residue conserved in most rhomboids that stabilizes the catalytic general base, to tyrosine, provided insight into the mechanism of nucleophile generation for the catalytic dyad. This study provides a quantitative evaluation of the role of several residues important for hydrolytic efficiency and oxyanion stabilization during intramembrane proteolysis.

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Comparative analysis of the molecular mechanism of heat shock proteins from pathogenic and non-pathogenic E.coli

10.1101/2020.06.17.157248 ◽

2020 ◽

Author(s):

Sanchari Bhattacharjee ◽

Mohana Saha ◽

Rakhi Dasgupta ◽

Angshuman Bagchi

Keyword(s):

Heat Shock ◽

Heat Shock Proteins ◽

Temperature Stress ◽

Small Heat Shock Proteins ◽

Lon Protease ◽

E Coli ◽

Physiological Temperature ◽

Mechanism Of Interaction ◽

Shock Proteins ◽

Catalytic Dyad

AbstractCells can withstand the effects of temperature stress by activating small heat shock proteins IbpA and IbpB. Lon protease employing Ser679 – Lys722 catalytic dyad proteolyze IbpA and IbpB in their free forms, at physiological temperature i.e. without any temperature stress. However, the proteolytic activity of IbpA and IbpB is affected when the catalytic dyad residue of Lon protease is mutated. The mutation S679A in Lon protease brings about some changes so that the proteolytic interactions between the small heat shock proteins with that of the mutant Lon protease are lost which makes a difference in the interaction pattern of mutant Lon protease with their substrates. In the present study, we made an attempt through in-silico approach to figure out the underlying aspects of the interactions between the small heat shock proteins IbpA and IbpB with mutant Lon protease in Escherichia coli. We have tried to decipher the molecular details of the mechanism of interaction of proteolytic machinery of small heat shock proteins and mutant Lon protease with S679A mutation at physiological temperature in absence cellular temperature stress. Our study may therefore be helpful to decode the mechanistic details of the correlation with IbpA, IbpB and S679A mutant Lon protease in E. coli.

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The Thermoplasma acidophilum Lon protease has a Ser-Lys dyad active site

European Journal of Biochemistry ◽

10.1111/j.1432-1033.2004.04421.x ◽

2004 ◽

Vol 271 (22) ◽

pp. 4361-4365 ◽

Cited By ~ 16

Author(s):

Henrike Besche ◽

Peter Zwickl

Keyword(s):

Active Site ◽

Lon Protease ◽

Thermoplasma Acidophilum

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Identification of Active-Site Amino Acid Residues in the Chiba Virus 3C-Like Protease

Journal of Virology ◽

10.1128/jvi.76.12.5949-5958.2002 ◽

2002 ◽

Vol 76 (12) ◽

pp. 5949-5958 ◽

Cited By ~ 37

Author(s):

Yuichi Someya ◽

Naokazu Takeda ◽

Tatsuo Miyamura

Keyword(s):

Amino Acid ◽

Active Site ◽

Hepatitis A Virus ◽

Side Chain ◽

Significant Activity ◽

Amino Acid Residues ◽

Active Site Residues ◽

3C Protease ◽

Catalytic Dyad ◽

Positively Charged

ABSTRACT The 3C-like protease of the Chiba virus, a Norwalk-like virus, is one of the chymotrypsin-like proteases. To identify active-site amino acid residues in this protease, 37 charged amino acid residues and a putative nucleophile, Cys139, within the GDCG sequence were individually replaced with Ala in the 3BC precursor, followed by expression in Escherichia coli, where the active 3C-like protease would cleave 3BC into 3B (VPg) and 3C (protease). Among 38 Ala mutants, 7 mutants (R8A, H30A, K88A, R89A, D138A, C139A, and H157A) completely or nearly completely lost the proteolytic activity. Cys139 was replaceable only with Ser, suggesting that an SH or OH group in the less bulky side chain was required for the side chain of the residue at position 139. His30, Arg89, and Asp138 could not be replaced with any other amino acids. Although Arg8 was also not replaceable for the 3B/3C cleavage and the 3C/3D cleavage, the N-terminal truncated mutant devoid of Arg8 significantly cleaved 3CD into 3C and 3D (polymerase), indicating that Arg8 itself was not directly involved in the proteolytic cleavage. As for position 88, a positively charged residue was required because the Arg mutant showed significant activity. As deduced by the X-ray structure of the hepatitis A virus 3C protease, Arg8, Lys88, and Arg89 are far away from the active site, and the side chain of Asp138 is directed away from the active site. Therefore, these are not catalytic residues. On the other hand, all of the mutants of His157 in the S1 specificity pocket tended to retain very slight activity, suggesting a decreased level of substrate recognition. These results, together with a sequence alignment with the picornavirus 3C proteases, indicate that His30 and Cys139 are active-site residues, forming a catalytic dyad without a carboxylate directly participating in the proteolysis.

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Active Site Mapping of an Aspartic Protease by Multiple Fragment Crystal Structures: Versatile Warheads To Address a Catalytic Dyad

Journal of Medicinal Chemistry ◽

10.1021/acs.jmedchem.6b01195 ◽

2016 ◽

Vol 59 (21) ◽

pp. 9743-9759 ◽

Cited By ~ 8

Author(s):

Nedyalka Radeva ◽

Johannes Schiebel ◽

Xiaojie Wang ◽

Stefan G. Krimmer ◽

Kan Fu ◽

...

Keyword(s):

Crystal Structures ◽

Active Site ◽

Aspartic Protease ◽

Site Mapping ◽

Catalytic Dyad ◽

Multiple Fragment

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In Silico Screening of Naturally Occurring Coumarin Derivatives for the Inhibition of the Main Protease of SARS-CoV-2

10.26434/chemrxiv.12234728 ◽

2020 ◽

Author(s):

Sona Lyndem ◽

Sharat Sarmah ◽

Sourav Das ◽

Atanu Singha Roy

Keyword(s):

Active Site ◽

Preliminary Screening ◽

Naturally Occurring ◽

Main Protease ◽

Coumarin Compounds ◽

Potential Inhibitors ◽

Novel Drug ◽

Catalytic Dyad

The dissemination of a novel corona virus, SARS-CoV-2, through rapid human to human transmission has led to a global health emergency. The lack of a vaccine or medication for effective treatment of this disease has made it imperative for developing novel drug discovery approaches. Repurposing of drugs is one such method currently being used to tackle the viral infection. The genome of SARS-CoV-2 replicates due to the functioning of a main protease called Mpro. By targeting the active site of Mpro with potential inhibitors, this could prevent viral replication from taking place. Blind docking technique was used to investigate the interactions between 29 naturally occurring coumarin compounds and SARS-CoV-2 main protease, Mpro, out of which 17 coumarin compounds were seen to bind to the active site through the interaction with the catalytic dyad, His41 and Cys145, along with other neighbouring residues. On comparing the ΔG values of the coumarins bound to the active site of Mpro, corymbocoumarin belonging to the class pyranocoumarins, methylgalbanate belonging to the class simple coumarins and heraclenol belonging to the class furanocoumarins, displayed best binding efficiency and could be considered as potential Mpro protease inhibitors. Preliminary screening of these naturally occurring coumarin compounds as potential SARS-CoV-2 replication inhibitors acts as a stepping stone for further in vitro and in vivo experimental investigation and analytical validation.

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Elucidation of the molecular mechanism of heat shock proteins and its correlation with the effects of double mutations S679A & K722Q in the catalytic dyad residues of Lon protease

Gene Reports ◽

10.1016/j.genrep.2019.100438 ◽

2019 ◽

Vol 16 ◽

pp. 100438 ◽

Cited By ~ 1

Author(s):

Sanchari Bhattacharjee ◽

Indranil Halder ◽

Sangeeta Mitra ◽

Rakhi Dasgupta ◽

Angshuman Bagchi

Keyword(s):

Heat Shock ◽

Heat Shock Proteins ◽

Molecular Mechanism ◽

Lon Protease ◽

Shock Proteins ◽

Catalytic Dyad

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The rhomboid protease GlpG has weak interaction energies in its active site hydrogen bond network

The Journal of General Physiology ◽

10.1085/jgp.201812047 ◽

2018 ◽

Vol 151 (3) ◽

pp. 282-291

Author(s):

Kristen A. Gaffney ◽

Heedeok Hong

Keyword(s):

Hydrogen Bonding ◽

Crystal Structures ◽

Weak Interaction ◽

Active Site ◽

Critical Role ◽

Interaction Energies ◽

Active Site Residues ◽

Rhomboid Protease ◽

Rhomboid Proteases ◽

Catalytic Dyad

Intramembrane rhomboid proteases are of particular interest because of their function to hydrolyze a peptide bond of a substrate buried in the membrane. Crystal structures of the bacterial rhomboid protease GlpG have revealed a catalytic dyad (Ser201-His254) and oxyanion hole (His150/Asn154/the backbone amide of Ser201) surrounded by the protein matrix and contacting a narrow water channel. Although multiple crystal structures have been solved, the catalytic mechanism of GlpG is not completely understood. Because it is a serine protease, hydrogen bonding interactions between the active site residues are thought to play a critical role in the catalytic cycle. Here, we dissect the interaction energies among the active site residues His254, Ser201, and Asn154 of Escherichia coli GlpG, which form a hydrogen bonding network. We combine double mutant cycle analysis with stability measurements using steric trapping. In mild detergent, the active site residues are weakly coupled with interaction energies (ΔΔGInter) of ‒1.4 kcal/mol between His254 and Ser201 and ‒0.2 kcal/mol between Ser201 and Asn154. Further, by analyzing the propagation of single mutations of the active site residues, we find that these residues are important not only for function but also for the folding cooperativity of GlpG. The weak interaction between Ser and His in the catalytic dyad may partly explain the unusually slow proteolysis by GlpG compared with other canonical serine proteases. Our result suggests that the weak hydrogen bonds in the active site are sufficient to carry out the proteolytic function of rhomboid proteases.

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Redundant In Vivo Proteolytic Activities ofEscherichia coli Lon and the ClpYQ (HslUV) Protease

Journal of Bacteriology ◽

10.1128/jb.181.12.3681-3687.1999 ◽

1999 ◽

Vol 181 (12) ◽

pp. 3681-3687 ◽

Cited By ~ 74

Author(s):

Whi-Fin Wu ◽

YanNing Zhou ◽

Susan Gottesman

Keyword(s):

Active Site ◽

Elevated Temperatures ◽

Lon Protease ◽

Uv Resistance ◽

Atpase Subunit ◽

Uv Sensitivity ◽

Proteolytic Activities ◽

Cell Division Inhibitor ◽

Single Subunit

ABSTRACT The ClpYQ (HslUV) ATP-dependent protease of Escherichia coli consists of an ATPase subunit closely related to the Clp ATPases and a protease component related to those found in the eukaryotic proteasome. We found that this protease has a substrate specificity overlapping that of the Lon protease, another ATP-dependent protease in which a single subunit contains both the proteolytic active site and the ATPase. Lon is responsible for the degradation of the cell division inhibitor SulA; lon mutants are UV sensitive, due to the stabilization of SulA. lon mutants are also mucoid, due to the stabilization of another Lon substrate, the positive regulator of capsule transcription, RcsA. The overproduction of ClpYQ suppresses both of these phenotypes, and the suppression of UV sensitivity is accompanied by a restoration of the rapid degradation of SulA. Inactivation of the chromosomal copy of clpY orclpQ leads to further stabilization of SulA in alon mutant but not in lon + cells. While either lon, lon clpY, or lon clpQ mutants are UV sensitive at low temperatures, at elevated temperatures the lon mutant loses its UV sensitivity, while the double mutants do not. Therefore, the degradation of SulA by ClpYQ at elevated temperatures is sufficient to lead to UV resistance. Thus, a protease with a structure and an active site different from those of Lon is capable of recognizing and degrading two different Lon substrates and appears to act as a backup for Lon under certain conditions.

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