scholarly journals Crystal structure of Mycobacterium tuberculosis FadB2 implicated in mycobacterial β-oxidation

2019 ◽  
Vol 75 (1) ◽  
pp. 101-108 ◽  
Author(s):  
Jonathan A. G. Cox ◽  
Rebecca C. Taylor ◽  
Alistair K. Brown ◽  
Samuel Attoe ◽  
Gurdyal S. Besra ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mycobacterium Tuberculosis ◽  
Critical Role ◽  
Structural Features ◽  
Free Form ◽  
Cofactor Binding ◽  
Terminal Domain ◽  
Hydroxyacyl Coa Dehydrogenase ◽  
Self Association ◽  
Human Orthologue

The intracellular pathogen Mycobacterium tuberculosis is the causative agent of tuberculosis, which is a leading cause of mortality worldwide. The survival of M. tuberculosis in host macrophages through long-lasting periods of persistence depends, in part, on breaking down host cell lipids as a carbon source. The critical role of fatty-acid catabolism in this organism is underscored by the extensive redundancy of the genes implicated in β-oxidation (∼100 genes). In a previous study, the enzymology of the M. tuberculosis L-3-hydroxyacyl-CoA dehydrogenase FadB2 was characterized. Here, the crystal structure of this enzyme in a ligand-free form is reported at 2.1 Å resolution. FadB2 crystallized as a dimer with three unique dimer copies per asymmetric unit. The structure of the monomer reveals a dual Rossmann-fold motif in the N-terminal domain, while the helical C-terminal domain mediates dimer formation. Comparison with the CoA- and NAD+-bound human orthologue mitochondrial hydroxyacyl-CoA dehydrogenase shows extensive conservation of the residues that mediate substrate and cofactor binding. Superposition with the multi-catalytic homologue M. tuberculosis FadB, which forms a trifunctional complex with the thiolase FadA, indicates that FadB has developed structural features that prevent its self-association as a dimer. Conversely, FadB2 is unable to substitute for FadB in the tetrameric FadA–FadB complex as it lacks the N-terminal hydratase domain of FadB. Instead, FadB2 may functionally (or physically) associate with the enoyl-CoA hydratase EchA8 and the thiolases FadA2, FadA3, FadA4 or FadA6 as suggested by interrogation of the STRING protein-network database.

scholarly journals Structural and functional analyses of the N-terminal domain of the A subunit of aBacillus megateriumspore germinant receptor

2019 ◽  
Vol 116 (23) ◽  
pp. 11470-11479 ◽  
Author(s):  
Yunfeng Li ◽  
Kai Jin ◽  
Abigail Perez-Valdespino ◽  
Kyle Federkiewicz ◽  
Andrew Davis ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Abc Transporters ◽  
Spore Germination ◽  
Critical Role ◽  
Chemical Shift Perturbation ◽  
Terminal Domain ◽  
Proposed Model ◽  
Strong Homology ◽  
A Subunit ◽  
Functional Analyses

Germination ofBacillusspores is induced by the interaction of specific nutrient molecules with germinant receptors (GRs) localized in the spore’s inner membrane. GRs typically consist of three subunits referred to as A, B, and C, although functions of individual subunits are not known. Here we present the crystal structure of the N-terminal domain (NTD) of the A subunit of theBacillus megateriumGerK3GR, revealing two distinct globular subdomains bisected by a cleft, a fold with strong homology to substrate-binding proteins in bacterial ABC transporters. Molecular docking, chemical shift perturbation measurement, and mutagenesis coupled with spore germination analyses support a proposed model that the interface between the two subdomains in the NTD of GR A subunits serves as the germinant binding site and plays a critical role in spore germination. Our findings provide a conceptual framework for understanding the germinant recruitment mechanism by which GRs trigger spore germination.

scholarly journals Crystal structure of the toxin Msmeg_6760, the structural homolog ofMycobacterium tuberculosisRv2035, a novel type II toxin involved in the hypoxic response

2016 ◽  
Vol 72 (12) ◽  
pp. 863-869 ◽  
Author(s):  
R. Alexandra Bajaj ◽  
Mark A. Arbing ◽  
Annie Shin ◽  
Duilio Cascio ◽  
Linda Miallau
Keyword(s):  
Crystal Structure ◽  
Mycobacterium Tuberculosis ◽  
Structural Features ◽  
Bacterial Toxins ◽  
Type Ii ◽  
Structural Comparison ◽  
Macrophage Infection ◽  
Bioinformatics Analyses ◽  
Hydrophobic Residues ◽  
Hydrophobic Ligand

The structure of Msmeg_6760, a protein of unknown function, has been determined. Biochemical and bioinformatics analyses determined that Msmeg_6760 interacts with a protein encoded in the same operon, Msmeg_6762, and predicted that the operon is a toxin–antitoxin (TA) system. Structural comparison of Msmeg_6760 with proteins of known function suggests that Msmeg_6760 binds a hydrophobic ligand in a buried cavity lined by large hydrophobic residues. Access to this cavity could be controlled by a gate–latch mechanism. The function of the Msmeg_6760 toxin is unknown, but structure-based predictions revealed that Msmeg_6760 and Msmeg_6762 are homologous to Rv2034 and Rv2035, a predicted novel TA system involved inMycobacterium tuberculosislatency during macrophage infection. The Msmeg_6760 toxin fold has not been previously described for bacterial toxins and its unique structural features suggest that toxin activation is likely to be mediated by a novel mechanism.

scholarly journals Structural characterization of the N-terminal domain of the Dictyostelium discoideum mitochondrial calcium uniporter

2019 ◽  
Author(s):  
Yuan Yuan ◽  
Chan Cao ◽  
Maorong Wen ◽  
Min Li ◽  
Ying Dong ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Dictyostelium Discoideum ◽  
Self Assembly ◽  
Calcium Influx ◽  
Transmembrane Protein ◽  
Critical Role ◽  
Mitochondrial Calcium ◽  
Mitochondrial Calcium Uniporter ◽  
Terminal Domain ◽  
Calcium Uniporter

AbstractThe mitochondrial calcium uniporter (MCU) plays a critical role in the mitochondrial calcium uptake into the matrix. In metazoans, the uniporter is a tightly regulated multi-component system including the pore-forming subunit MCU and several regulators (MICU1, MICU2, EMRE). The calcium-conducting activity of metazoan MCU requires the single-transmembrane protein EMRE. Dictyostelium discoideum (Dd), however, developed a simplified uniporter for which the pore-forming MCU (DdMCU) alone is necessary and sufficient for calcium influx. Here, we report a crystal structure of the N-terminal domain (NTD) of DdMCU at 1.7 Å resolution. The DdMCU-NTD contains four helices and two strands arranged in a fold that is completely different from the known structures of other MCU-NTD homologs. Biochemical and biophysical analyses of DdMCU-NTD in solution indicated that the domain exists as oligomers, most probably as a pentamer or hexamer. Mutagenesis showed that the acidic residues Asp60, Glu72 and Glu74, which appeared to mediate the parallel interface as observed in the crystal structure, participated in the self-assembly of DdMCU-NTD. Intriguingly, the oligomeric complex readily dissociated to lower-order oligomers in the presence of calcium. We propose that the calcium-triggered dissociation of NTD regulates the channel activity of DdMCU by a yet unknown mechanism.

scholarly journals Structure of the Paramyxovirus Parainfluenza Virus 5 Nucleoprotein in Complex with an Amino-Terminal Peptide of the Phosphoprotein

2017 ◽  
Vol 92 (5) ◽  
Author(s):  
Megha Aggarwal ◽  
George P. Leser ◽  
Christopher A. Kors ◽  
Robert A. Lamb
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Rna Binding ◽  
Hinge Region ◽  
Parainfluenza Virus ◽  
Free Form ◽  
Conformational Switching ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Parainfluenza Virus 5

ABSTRACT Parainfluenza virus 5 (PIV5) belongs to the family Paramyxoviridae , which consists of enveloped viruses with a nonsegmented negative-strand RNA genome encapsidated by the nucleoprotein (N). Paramyxovirus replication is regulated by the phosphoprotein (P) through protein-protein interactions with N and the RNA polymerase (L). The chaperone activity of P is essential to maintain the unassembled RNA-free form of N in order to prevent nonspecific RNA binding and premature N oligomerization. Here, we determined the crystal structure of unassembled PIV5 N in complex with a P peptide (N 0 P) derived from the N terminus of P (P50) at 2.65 Å. The PIV5 N 0 P consists of two domains: an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a hinge region. The cleft at the hinge region of RNA-bound PIV5 N was previously shown to be an RNA binding site. The N 0 P structure shows that the P peptide binds to the CTD of N and extends toward the RNA binding site to inhibit N oligomerization and, hence, RNA binding. Binding of P peptide also keeps the PIV5 N in the open form. A molecular dynamics (MD) analysis of both the open and closed forms of N shows the flexibility of the CTD and the preference of the N protein to be in an open conformation. The gradual opening of the hinge region, to release the RNA, was also observed. Together, these results advance our knowledge of the conformational swapping of N required for the highly regulated paramyxovirus replication. IMPORTANCE Paramyxovirus replication is regulated by the interaction of P with N and L proteins. Here, we report the crystal structure of unassembled parainfluenza virus 5 (PIV5) N chaperoned with P peptide. Our results provide a detailed understanding of the binding of P to N. The conformational switching of N between closed and open forms during its initial interaction with P, as well as during RNA release, was analyzed. Our data also show the plasticity of the CTD and the importance of domain movement for conformational switching. The results improve our understanding of the mechanism of interchanging N conformations for RNA replication and release.

scholarly journals Regulation of polyphosphate glucokinase gene expression through co-transcriptional processing in Mycobacterium tuberculosis H37Rv

2020 ◽  
Author(s):  
Naveen Prakash Bokolia ◽  
Inshad Ali Khan
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Critical Role ◽  
Structural Features ◽  
Mrna Processing ◽  
Coding Region ◽  
Rna Molecules ◽  
Mycobacterium Tuberculosis H37rv ◽  
Cleavage Activity ◽  
Cellular Environment ◽  
Nascent Rna

AbstractTranscription is the process that allows the simultaneous folding of RNA molecules, known as co-transcriptional folding. This folding determines the functional properties of RNA molecules and possibly having a critical role during the synthesis as well. This functioning includes the characterized properties of riboswitches and ribozymes as well, which is significant when the transcription rate is comparable to the cellular environment. This study aims to discover a novel non-coding region that is important in the genetic expression of Mycobacterium tuberculosis. In this work, we identified a novel non-coding element of polyphosphate glucokinase (ppgk) gene that undergoes cleavage activity during the transcriptional process in Mycobacterium tuberculosis. We revealed that cleavage occurs within the nascent RNA, and the resultant cleaved 3’RNA fragment carries the Shine-Dalgarno (SD) sequence and expression platform. Site-specific mutations provide a strong correlation between the disruption of cleavage activity and expression of ppgk gene. We concluded that co-transcriptional processing at the noncoding region as the required mechanism for ppgk expression that remains constitutive within the bacterial environment. The underlying reason for ppgk mRNA processing and expression is correlated because the non-coding counterpart adopts a hairpin domain that sequesters ribosomal binding site. Thus, the mRNA processing at the immediate upstream of Shine-Dalgarno sequence is required to prevent this sequestration and subsequent expression as well. This study defines the molecular mechanism that is dependent on the transient but highly active structural features of the nascent RNA.

scholarly journals Structural Insights into HIV-1 Vif-APOBEC3F Interaction

2015 ◽  
Vol 90 (2) ◽  
pp. 1034-1047 ◽  
Author(s):  
Masaaki Nakashima ◽  
Hirotaka Ode ◽  
Takashi Kawamura ◽  
Shingo Kitamura ◽  
Yuriko Naganawa ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Hydrophobic Interactions ◽  
Structural Features ◽  
Sequence Motifs ◽  
Structural Mechanism ◽  
Terminal Domain ◽  
Interaction Interface ◽  
Important Basis ◽  
Structural Insights ◽  
Hiv 1

ABSTRACTThe HIV-1 Vif protein inactivates the cellular antiviral cytidine deaminase APOBEC3F (A3F) in virus-infected cells by specifically targeting it for proteasomal degradation. Several studies identified Vif sequence motifs involved in A3F interaction, whereas a Vif-binding A3F interface was proposed based on our analysis of highly similar APOBEC3C (A3C). However, the structural mechanism of specific Vif-A3F recognition is still poorly understood. Here we report structural features of interaction interfaces for both HIV-1 Vif and A3F molecules. Alanine-scanning analysis of Vif revealed that six residues located within the conserved Vif F1-, F2-, and F3-box motifs are essential for both A3C and A3F degradation, and an additional four residues are uniquely required for A3F degradation. Modeling of the Vif structure on an HIV-1 Vif crystal structure revealed that three discontinuous flexible loops of Vif F1-, F2-, and F3-box motifs sterically cluster to form a flexible A3F interaction interface, which represents hydrophobic and positively charged surfaces. We found that the basic Vif interface patch (R17, E171, and R173) involved in the interactions with A3C and A3F differs. Furthermore, our crystal structure determination and extensive mutational analysis of the A3F C-terminal domain demonstrated that the A3F interface includes a unique acidic stretch (L291, A292, R293, and E324) crucial for Vif interaction, suggesting additional electrostatic complementarity to the Vif interface compared with the A3C interface. Taken together, these findings provide structural insights into the A3F-Vif interaction mechanism, which will provide an important basis for development of novel anti-HIV-1 drugs using cellular cytidine deaminases.IMPORTANCEHIV-1 Vif targets cellular antiviral APOBEC3F (A3F) enzyme for degradation. However, the details on the structural mechanism for specific A3F recognition remain unclear. This study reports structural features of interaction interfaces for both HIV-1 Vif and A3F molecules. Three discontinuous sequence motifs of Vif, F1, F2, and F3 boxes, assemble to form an A3F interaction interface. In addition, we determined a crystal structure of the wild-type A3F C-terminal domain responsible for the Vif interaction. These results demonstrated that both electrostatic and hydrophobic interactions are the key force driving Vif-A3F binding and that the Vif-A3F interfaces are larger than the Vif-A3C interfaces. These findings will allow us to determine the configurations of the Vif-A3F complex and to construct a structural model of the complex, which will provide an important basis for inhibitor development.

scholarly journals Crystal structure of the DNA gyrase GyrA N-terminal domain from Mycobacterium tuberculosis

2009 ◽  
Vol 78 (2) ◽  
pp. 492-495 ◽  
Author(s):  
Elsa M. Tretter ◽  
Allyn J. Schoeffler ◽  
Shellie R. Weisfield ◽  
James M. Berger
Keyword(s):  
Crystal Structure ◽  
Mycobacterium Tuberculosis ◽  
Dna Gyrase ◽  
Terminal Domain

scholarly journals Crystal Structure and Activity Studies of the Mycobacterium tuberculosis β-Lactamase Reveal Its Critical Role in Resistance to β-Lactam Antibiotics

2006 ◽  
Vol 50 (8) ◽  
pp. 2762-2771 ◽  
Author(s):  
Feng Wang ◽  
Craig Cassidy ◽  
James C. Sacchettini
Keyword(s):  
Crystal Structure ◽  
Mycobacterium Tuberculosis ◽  
Critical Role ◽  
Gram Negative Bacteria ◽  
Gene Encoding ◽  
Class A ◽  
Increased Sensitivity ◽  
Distinct Features ◽  
Structure And Activity

ABSTRACT β-Lactam antibiotics are extremely effective in disrupting the synthesis of the bacterial cell wall in both gram-positive and gram-negative bacteria. However, they are ineffective against Mycobacterium tuberculosis, due to the production of a β-lactamase enzyme encoded on the chromosome of M. tuberculosis that degrades these antibiotics. Indeed, recent studies have demonstrated that deletion of the blaC gene, the only gene encoding a β-lactamase in M. tuberculosis, or inhibition of the encoded enzyme resulted in significantly increased sensitivity to β-lactam antibiotics. In this paper we present a biochemical and structural characterization of M. tuberculosis BlaC. Recombinant BlaC shows a broad range of specificity with almost equal penicillinase and cepholothinase activity. While clavulanate is a mechanism-based inhibitor to class A β-lactamase with high potency (typically Ki < 0.1 μM), it is a relatively poor inhibitor of the M. tuberculosis BlaC (Ki = 2.4 μM). The crystal structure of the enzyme, determined at a resolution of 1.7 Å, shows that the overall fold of the M. tuberculosis enzyme is similar to other class A β-lactamases. There are, however, several distinct features of the active site, such as the amino acid substitutions N132G, R164A, R244A, and R276E, that explain the broad specificity of the enzyme, relatively low penicillinase activity, and resistance to clavulanate.

Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

2019 ◽  
Vol 476 (21) ◽  
pp. 3227-3240 ◽  
Author(s):  
Shanshan Wang ◽  
Yanxiang Zhao ◽  
Long Yi ◽  
Minghe Shen ◽  
Chao Wang ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Magnaporthe Oryzae ◽  
Structural Information ◽  
Complex Structure ◽  
Critical Role ◽  
Catalytic Process ◽  
Structural Basis ◽  
Salt Bridges ◽  
Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

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