Biology subjects

Minasov, G.

Publications and source records attributed to Minasov, G..

9 recordsLinked to original sources

bioRxiv2026

Control of cell division by an Acinetobacter baumannii protein with a novel nucleotidyl-cyclase-like fold

The antibiotic-resistant pathogen Acinetobacter baumannii has diverged from model {psi}-proteobacteria in fundamental ways, complicating the development of new antimicrobial strategies. A major area of divergence is cell division. A. baumannii lacks several widely conserved division enzymes, such as FtsEX, and instead possesses a suite of atypical gene products with no similarity to well-characterized proteins. Key among these is AdvA, which we previously identified by Tn-seq as essential for A. baumannii division and fluoroquinolone resistance. The protein comprises an N-terminal transmembrane/periplasmic region connected to a C-terminal unannotated cytoplasmic domain, and most advA transposon insertions were lethal unless they occurred within the linker between these regions. The roles of AdvA in cell division and the basis for these positional transposon effects were unclear. Here, we combine mutagenesis with fluorescence localization, two-hybrid, and structural analyses to define how AdvA domains function in assembling and activating the A. baumannii divisome. AdvA depletion profoundly disrupts divisome construction at Z-rings. This dependence reflects numerous interactions with divisome proteins, with AdvAs N-terminal region binding multiple components and cytoplasmic domain binding one, the early protein ZipA. In addition, we identified substitutions in FtsB and FtsW that suppress AdvA essentiality, consistent with a role in divisome activation as well as recruitment. Finally, we determined the structure of the cytoplasmic domain, revealing a novel adenylyl/guanylyl cyclase-like fold that lacks canonical catalytic and dimerization sites and instead features a positively charged tip key to fluoroquinolone resistance and a C-terminal helix essential to division. The critical C-terminal structure helps explain the positional transposon effects and facilitated identification of a distant homolog in Pseudomonas aeruginosa. These results reveal a new control protein governing bacterial division that could be exploited to combat nosocomial infections. ImportanceThe multidrug-resistant sepsis pathogen Acinetobacter baumannii poses an urgent threat to public health. Fundamental features of its cell cycle, such as how it controls cell division, are not well understood, but this information could lead to improved antimicrobial strategies. We demonstrate that a protein (AdvA) bearing a previously unrecognized structure has a critical role in cell division and fluoroquinolone antibiotic resistance in the pathogen. The structure resembles the nucleotide cyclase class of enzymes, but it has lost the typical catalytic properties and instead uses novel sites to enable assembly and activation of the cell division machine. The novel fold is found in other pathogens such as Pseudomonas aeruginosa, in which it is also connected to drug resistance and cell division. This work opens new avenues to understand and interrupt cell division in multidrug-resistant hospital-acquired bacteria.

microbiology↗

bioRxiv2024

Torsional Twist of the SARS-CoV and SARS-CoV-2 SUD-N and SUD-M domains

Coronavirus non-structural protein 3 (nsp3) forms hexameric crowns of pores in the double membrane vacuole that houses the replication-transcription complex. Nsp3 in SARS-like viruses has three unique domains absent in other coronavirus nsp3 proteins. Two of these, SUD-N (Macrodomain 2) and SUD-M (Macrodomain 3), form two lobes connected by a peptide linker and an interdomain disulfide bridge. We resolve the first complete x-ray structure of SARS-CoV SUD-N/M as well as a mutant variant of SARS-CoV-2 SUD-N/M modified to restore cysteines for interdomain disulfide bond naturally lost by evolution. Comparative analysis of all structures revealed SUD-N and SUD-M are not rigidly associated, but rather, have significant rotational flexibility. Phylogenetic analysis supports that the disulfide bond cysteines are also absent in pangolin-SARS and closely related viruses, consistent with pangolins being the presumed intermediate host in the emergence of SARS-CoV-2. The absence of these cysteines does not impact viral replication or protein translation.

microbiology↗

bioRxiv2023

Vibrio MARTX toxin processing and degradation of cellular Rab GTPases by the cytotoxic effector Makes Caterpillars Floppy

Vibrio vulnificus causes life threatening infections dependent upon the effectors released from the Multifunctional-Autoprocessing Repeats-In-Toxin (MARTX) toxin. The Makes Caterpillars Floppy-like (MCF) cysteine protease effector is activated by host ADP ribosylation factors (ARFs), although the targets of processing activity were unknown. In this study we show MCF binds Ras-related proteins in brain (Rab) GTPases at the same interface occupied by ARFs and then cleaves and/or degrades 24 distinct members of the Rab GTPases family. The cleavage occurs in the C-terminal tails of Rabs. We determine the crystal structure of MCF as a swapped dimer revealing the open, activated state of MCF and then use structure prediction algorithms to show that structural composition, rather than sequence or localization, determine Rabs selected as MCF proteolytic targets. Once cleaved, Rabs become dispersed in cells to drive organelle damage and cell death to promote pathogenesis of these rapidly fatal infections.

microbiology↗

bioRxiv2022

Structure of the Monkeypox profilin-like protein A42R reveals potential function differences from cellular profilins

The infectious disease human monkeypox is spreading rapidly in 2022 causing a global health crisis. The genomics of the monkeypox virus (MPXV) have been extensively analyzed and reported, although little is known about the virus-encoded proteome. In particular, there is almost no structure information about MPXV proteins, other than computational models. Here we report a 1.52 [A] x-ray structure of the MPXV protein A42R, the only MPXV-encoded protein with a known structure. This protein shows sequence similarity to profilins, which are cellular proteins known to function in regulation of actin cytoskeletal assembly. However, structural comparison of the A42R with known members of the profilin family reveals critical differences that support prior biochemical findings that this protein only weakly binds actin and does not bind poly(L-proline). In addition, the analysis suggests that this protein may have distinct interactions with phosphatidylinositol lipids. Overall, our data suggest that the role of A42R in replication of orthopoxviruses may not be easily determined by comparison to cellular profilins. Further, our findings support a need for increased efforts to determine high-resolution structures of other MPXV proteins to inform physiological studies of the poxvirus infection cycle and to reveal potential new strategies to combat human monkeypox should the infection become more common in the future.

microbiology↗

bioRxiv2021

Mn2+ coordinates Cap-0-RNA to align substrates for efficient 2'-O-methyl transfer by SARS-CoV-2 nsp16

Capping viral messenger RNAs is essential for efficient translation and prevents their detection by host innate immune responses. For SARS-CoV-2, RNA capping includes 2'-O-methylation of the first ribonucleotide by methyltransferase nsp16 in complex with activator nsp10. The reaction requires substrates, a short RNA and SAM, and is catalyzed by divalent cations, with preference for Mn2+. Crystal structures of nsp16-nsp10 with capped RNAs revealed a critical role of metal ions in stabilizing interactions between ribonucleotides and nsp16, resulting in precise alignment of the substrates for methyl transfer. An aspartate residue that is highly conserved among coronaviruses alters the backbone conformation of the capped RNA in the binding groove. This aspartate is absent in mammalian methyltransferases and is a promising site for designing coronavirus-specific inhibitors.

microbiology↗

bioRxiv2020

The ChiS family DNA-binding domain contains a cryptic helix-turn-helix variant

Sequence specific DNA-binding domains (DBDs) are conserved in all domains of life. These proteins carry out a variety of cellular functions, and there are a number of distinct structural domains already described that allow for sequence-specific DNA binding, including the ubiquitous helix-turn-helix (HTH) domain. In the facultative pathogen Vibrio cholerae, the chitin sensor ChiS is a transcriptional regulator that is critical for the survival of this organism in its marine reservoir. We have recently shown that ChiS contains a cryptic DBD in its C-terminus. This domain is not homologous to any known DBD, but it is a conserved domain present in other bacterial proteins. Here, we present the crystal structure of the ChiS DBD at a resolution of 1.28 [A]. We find that the ChiS DBD contains an HTH domain that is structurally similar to those found in other DNA binding proteins, like the LacI repressor. However, one striking difference observed in the ChiS DBD is that the canonical tight "turn" of the HTH is replaced with an insertion containing a {beta}-sheet, a variant which we term the "helix-sheet-helix". Through systematic mutagenesis of all positively charged residues within the ChiS DBD, we show that residues within and proximal to the ChiS helix-sheet-helix are critical for DNA binding. Finally, through phylogenetic analyses we show that the ChiS DBD is found in diverse Proteobacterial proteins that exhibit distinct domain architectures. Together, these results suggest that the structure described here represents the prototypical member of the ChiS-family of DBDs. ImportanceRegulating gene expression is essential in all domains of life. This process is commonly facilitated by the activity of DNA-binding transcription factors. There are diverse structural domains that allow proteins to bind to specific DNA sequences. The structural basis underlying how some proteins bind to DNA, however, remains unclear. Previously, we showed that in the major human pathogen Vibrio cholerae the transcription factor ChiS directly regulates gene expression through a cryptic DNA binding domain. This domain lacked homology to any known DNA-binding protein. In the current study, we determined the structure of the ChiS DNA binding domain (DBD) and find that the ChiS-family DBD is a cryptic variant of the ubiquitous helix-turn-helix (HTH) domain. We further demonstrate that this domain is conserved in diverse proteins that may represent a novel group of transcriptional regulators.

microbiology↗

bioRxiv2020

Structure of SARS-CoV-2 2'-O-methyltransferase heterodimer with RNA Cap analog and sulfates bound reveals new strategies for structure-based inhibitor design

There are currently no antiviral therapies specific against SARS-CoV-2, the virus responsible for the global pandemic disease COVID-19. To facilitate structure-based drug design, we conducted an X-ray crystallographic study of the nsp16/nsp10 2'-O-methyltransferase complex that methylates Cap-0 viral mRNAs to improve viral protein translation and to avoid host immune detection. Heterodimer structures are determined with the methyl donor S-adenosylmethionine (SAM), the reaction product S-adenosylhomocysteine (SAH) or the SAH analog sinefungin (SFG). Furthermore, structures of nsp16/nsp10 with the methylated Cap-0 analog (m7GpppA) and SAM or SAH bound were obtained. Comparative analysis revealed flexible loops in open and closed conformations at the m7GpppA binding pocket. Bound sulfates in several structures suggested the location of the phosphates in the ribonucleotide binding groove. Additional nucleotide binding sites were found on the face of the protein opposite the active site. These various sites and the conserved dimer interface could be exploited for development of antiviral inhibitors.

microbiology↗

bioRxiv2020

The crystal structure of nsp10-nsp16 heterodimer from SARS CoV-2in complex with S-adenosylmethionine

SARS-CoV-2 is a member of the coronaviridae family and is the etiological agent of the respiratory Coronavirus Disease 2019. The virus has spread rapidly around the world resulting in over two million cases and nearly 150,000 deaths as of April 17, 2020. Since no treatments or vaccines are available to treat COVID-19 and SARS-CoV-2, respiratory complications derived from the infections have overwhelmed healthcare systems around the world. This virus is related to SARS-CoV-1, the virus that caused the 2002-2004 outbreak of Severe Acute Respiratory Syndrome. In January 2020, the Center for Structural Genomics of Infectious Diseases implemented a structural genomics pipeline to solve the structures of proteins essential for coronavirus replication-transcription. Here we show the first structure of the SARS-CoV-2 nsp10-nsp16 2-O-methyltransferase complex with S-adenosylmethionine at a resolution of 1.80 [A]. This heterodimer complex is essential for capping viral mRNA transcripts for efficient translation and to evade immune surveillance.

biochemistry↗

bioRxiv2019

The Cyclic AMP Receptor Protein Regulates Quorum Sensing and Global Gene Expression in Yersinia pestis During Planktonic Growth and Growth in Biofilms

Cyclic adenosine monophosphate (cAMP) receptor protein (Crp) is an important transcriptional regulator of Yersinia pestis. Expression of crp increases during pneumonic plague as the pathogen depletes glucose and forms large biofilms within lungs. To better understand control of Y. pestis Crp, we determined a 1.8 [A] crystal structure of the protein-cAMP complex. We found that compared to Escherichia coli Crp, C helix amino acid substitutions in Y. pestis Crp did not impact cAMP dependency of Crp to bind DNA promoters. To investigate Y. pestis Crp-regulated genes during plague pneumonia, we performed RNA-sequencing on both wild-type and {Delta}crp mutant bacteria growing in planktonic and biofilm states in minimal media with glucose or glycerol. Y. pestis Crp is found to dramatically alter expression of hundreds of genes dependent upon carbon source and growth state. Gel shift assays confirmed direct regulation of the malT and ptsG promoters and Crp was then linked to Y. pestis growth on maltose as a sole carbon source. Iron-regulation genes ybtA and fyuA were found to be indirectly regulated by Crp. A new connection between carbon source and quorum sensing was revealed as Crp was found to regulate production of acyl-homoserine lactones (AHLs) through direct and indirect regulation of genes for AHL synthetases and receptors. AHLs were subsequently identified in the lungs of Y. pestis infected mice when crp expression is highest in Y. pestis biofilms. Thus, in addition to well-studied pla, other Crp-regulated genes likely have important functions during plague infection.\n\nIMPORTANCEBacterial pathogens have evolved extensive signaling pathways to translate environmental signals into changes in gene expression. While Crp has long been appreciated for its role in regulating metabolism of carbon sources in many bacterial species, transcriptional profiling has revealed that this protein regulates many other aspects of bacterial physiology. The plague pathogen, Y. pestis, requires this global regulator to survive in blood, skin, and lungs. During disease progression, this organism adapts to changes within these niches. In addition to regulating genes for metabolism of non-glucose sugars, we find the Crp regulates genes for virulence, metal acquisition and quorum sensing by direct or indirect mechanisms. Thus, this single transcriptional regulator, that responds to changes in available carbon sources, can regulate multiple critical behaviors for causing disease.

microbiology↗