Crystal structure of the complex of the interaction domains of Escherichia coli DnaB helicase and DnaC helicase loader: structural basis implying a distortion-accumulation mechanism for the DnaB ring opening caused by DnaC binding

2019 ◽  
Vol 167 (1) ◽  
pp. 1-14
Author(s):  
Koji Nagata ◽  
Akitoshi Okada ◽  
Jun Ohtsuka ◽  
Takatoshi Ohkuri ◽  
Yusuke Akama ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Molecular Model ◽  
Structural Basis ◽  
Terminal Domain ◽  
Helicase Dnab ◽  
Accumulation Mechanism ◽  
Interaction Domains ◽  
Α Helix ◽  
Available Information

Abstract Loading the bacterial replicative helicase DnaB onto DNA requires a specific loader protein, DnaC/DnaI, which creates the loading-competent state by opening the DnaB hexameric ring. To understand the molecular mechanism by which DnaC/DnaI opens the DnaB ring, we solved 3.1-Å co-crystal structure of the interaction domains of Escherichia coli DnaB–DnaC. The structure reveals that one N-terminal domain (NTD) of DnaC interacts with both the linker helix of a DnaB molecule and the C-terminal domain (CTD) of the adjacent DnaB molecule by forming a three α-helix bundle, which fixes the relative orientation of the two adjacent DnaB CTDs. The importance of the intermolecular interface in the crystal structure was supported by the mutational data of DnaB and DnaC. Based on the crystal structure and other available information on DnaB–DnaC structures, we constructed a molecular model of the hexameric DnaB CTDs bound by six DnaC NTDs. This model suggested that the binding of a DnaC would cause a distortion in the hexameric ring of DnaB. This distortion of the DnaB ring might accumulate by the binding of up to six DnaC molecules, resulting in the DnaB ring to open.

scholarly journals Crystal Structure of Classical Swine Fever Virus NS5B Reveals a Novel N-Terminal Domain

2018 ◽  
Vol 92 (14) ◽  
Author(s):  
Weiwei Li ◽  
Baixing Wu ◽  
Wibowo Adian Soca ◽  
Lei An
Keyword(s):  
Crystal Structure ◽  
Rna Polymerase ◽  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Structural Basis ◽  
Economic Losses ◽  
Rna Dependent Rna Polymerase ◽  
Content Type ◽  
Terminal Domain

ABSTRACTClassical swine fever virus (CSFV) is the cause of classical swine fever (CSF). Nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) that is a key enzyme initiating viral RNA replication by ade novomechanism. It is also an attractive target for the development of anti-CSFV drugs. To gain a better understanding of the mechanism of CSFV RNA synthesis, here, we solved the first crystal structure of CSFV NS5B. Our studies show that the CSFV NS5B RdRp contains the characteristic finger, palm, and thumb domains, as well as a unique N-terminal domain (NTD) that has never been observed. Mutagenesis studies on NS5B validated the importance of the NTD in the catalytic activity of this novel RNA-dependent RNA polymerase. Moreover, our results shed light on CSFV infection.IMPORTANCEPigs are important domesticated animals. However, a highly contagious viral disease named classical swine fever (CSF) causes devastating economic losses. Classical swine fever virus (CSFV), the primary cause of CSF, is a positive-sense single-stranded RNA virus belonging to the genusPestivirus, familyFlaviviridae. Genome replication of CSFV depends on an RNA-dependent RNA polymerase (RdRp) known as NS5B. However, the structure of CSFV NS5B has never been reported, and the mechanism of CSFV replication is poorly understood. Here, we solve the first crystal structure of CSFV NS5B and analyze the functions of the characteristic finger, palm, and thumb domains. Additionally, our structure revealed the presence of a novel N-terminal domain (NTD). Biochemical studies demonstrated that the NTD of CSFV NS5B is very important for RdRp activity. Collectively, our studies provide a structural basis for future rational design of anti-CSFV drugs, which is critically important, as no effective anti-CSFV drugs have been developed.

Structural basis for the interaction of BamB with the POTRA3–4 domains of BamA

2016 ◽  
Vol 72 (2) ◽  
pp. 236-244 ◽  
Author(s):  
Zhen Chen ◽  
Li-Hong Zhan ◽  
Hai-Feng Hou ◽  
Zeng-Qiang Gao ◽  
Jian-Hua Xu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Outer Membrane ◽  
Essential Role ◽  
Biochemical Analysis ◽  
Hydrophobic Interactions ◽  
Outer Membrane Proteins ◽  
Structural Basis ◽  
Conserved Residues ◽  
Bam Complex

InEscherichia coli, the Omp85 protein BamA and four lipoproteins (BamBCDE) constitute the BAM complex, which is essential for the assembly and insertion of outer membrane proteins into the outer membrane. Here, the crystal structure of BamB in complex with the POTRA3–4 domains of BamA is reported at 2.1 Å resolution. Based on this structure, the POTRA3 domain is associated with BamBviahydrogen-bonding and hydrophobic interactions. Structural and biochemical analysis revealed that the conserved residues Arg77, Glu127, Glu150, Ser167, Leu192, Leu194 and Arg195 of BamB play an essential role in interaction with the POTRA3 domain.

scholarly journals Family D DNA polymerase interacts with GINS to promote CMG-helicase in the archaeal replisome

2021 ◽  
Author(s):  
Keisuke Oki ◽  
Mariko Nagata ◽  
Takeshi Yamagami ◽  
Tomoyuki Numata ◽  
Sonoko Ishino ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Dna Polymerase ◽  
Direct Interaction ◽  
Crystal Structure Analysis ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Functional Connection ◽  
Terminal Domain ◽  
The Family ◽  
Lagging Strand

Abstract Genomic DNA replication requires replisome assembly. We show here the molecular mechanism by which CMG (GAN–MCM–GINS)-like helicase cooperates with the family D DNA polymerase (PolD) in Thermococcus kodakarensis. The archaeal GINS contains two Gins51 subunits, the C-terminal domain of which (Gins51C) interacts with GAN. We discovered that Gins51C also interacts with the N-terminal domain of PolD’s DP1 subunit (DP1N) to connect two PolDs in GINS. The two replicases in the replisome should be responsible for leading- and lagging-strand synthesis, respectively. Crystal structure analysis of the DP1N–Gins51C–GAN ternary complex was provided to understand the structural basis of the connection between the helicase and DNA polymerase. Site-directed mutagenesis analysis supported the interaction mode obtained from the crystal structure. Furthermore, the assembly of helicase and replicase identified in this study is also conserved in Eukarya. PolD enhances the parental strand unwinding via stimulation of ATPase activity of the CMG-complex. This is the first evidence of the functional connection between replicase and helicase in Archaea. These results suggest that the direct interaction of PolD with CMG-helicase is critical for synchronizing strand unwinding and nascent strand synthesis and possibly provide a functional machinery for the effective progression of the replication fork.

scholarly journals The Structure and Function of Modular Escherichia coli O157:H7 Bacteriophage FTBEc1 endolysin, LysT84: Defining a New Endolysin Catalytic Subfamily

2021 ◽  
Author(s):  
Michael Love ◽  
David Coombes ◽  
Salim Ismail ◽  
Craig Billington ◽  
Renwick CJ Dobson
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Antibacterial Agents ◽  
Analytical Ultracentrifugation ◽  
Structural Data ◽  
Scattering Data ◽  
Structural Basis ◽  
Escherichia Coli O157 ◽  
Dynamic Simulations ◽  
Enzymatic Function

Bacteriophage endolysins degrade peptidoglycan and have been identified as antibacterial candidates to combat antimicrobial resistance. Considering the catalytic and structural diversity of endolysins, there is a paucity of structural data to inform how these enzymes work at the molecular level—key data that is needed to realize the potential of endolysin-based antibacterial agents. Here, we determine the atomic structure and define the enzymatic function of Escherichia coli O157:H7 phage FTEBc1 endolysin, LysT84. Bioinformatic analysis reveals that LysT84 is a modular endolysin, which is unusual for Gram-negative endolysins, comprising a peptidoglycan binding domain and an enzymatic domain. The crystal structure of LysT84 (2.99 Å) revealed a mostly α-helical protein with two domains connected by a linker region but packed together. LysT84 was determined to be a monomer in solution using analytical ultracentrifugation. Small-angle X-ray scattering data revealed that LysT84 is a flexible protein but does not have the expected bimodal P(r) function of a multidomain protein, suggesting that the domains of LysT84 pack closely creating a globular protein as seen in the crystal structure. Structural analysis reveals two key glutamate residues positioned on either side of the active site cavity; mutagenesis demonstrating these residues are critical for peptidoglycan degradation. Molecular dynamic simulations suggest that the enzymatically active domain is dynamic, allowing the appropriate positioning of these catalytic residues for hydrolysis of the β(1–4) bond. Overall, our study defines the structural basis for peptidoglycan degradation by LysT84 which supports rational engineering of related endolysins into effective antibacterial agents.

scholarly journals Crystal Structure of the YchF Protein Reveals Binding Sites for GTP and Nucleic Acid

2003 ◽  
Vol 185 (14) ◽  
pp. 4031-4037 ◽  
Author(s):  
Alexey Teplyakov ◽  
Galina Obmolova ◽  
Seung Y. Chu ◽  
John Toedt ◽  
Edward Eisenstein ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Nucleic Acid ◽  
Binding Site ◽  
Coiled Coil ◽  
Genomic Analysis ◽  
Protein Fluorescence ◽  
Terminal Domain ◽  
Fluorescence Measurements ◽  
Α Helix

ABSTRACT The bacterial protein encoded by the gene ychF is 1 of 11 universally conserved GTPases and the only one whose function is unknown. The crystal structure determination of YchF was sought to help with the functional assignment of the protein. The YchF protein from Haemophilus influenzae was cloned and expressed, and the crystal structure was determined at 2.4 Å resolution. The polypeptide chain is folded into three domains. The N-terminal domain has a mononucleotide binding fold typical for the P-loop NTPases. An 80-residue domain next to it has a pronounced α-helical coiled coil. The C-terminal domain features a six-stranded half-barrel that curves around an α-helix. The crablike three-domain structure of YchF suggests the binding site for a double-stranded nucleic acid in the cleft between the domains. The structure of the putative GTP-binding site is consistent with the postulated guanine specificity of the protein. Fluorescence measurements have demonstrated the ability of YchF to bind a double-stranded nucleic acid and GTP. Taken together with other experimental data and genomic analysis, these results suggest that YchF may be part of a nucleoprotein complex and may function as a GTP-dependent translation factor.

scholarly journals Crystal structure of the N-terminal domain of the fibronectin-binding protein PavA from Streptococcus pneumoniae

2019 ◽  
Vol 75 (10) ◽  
pp. 657-662 ◽  
Author(s):  
Kartik Manne ◽  
Sthanam V. L. Narayana ◽  
Debasish Chattopadhyay
Keyword(s):  
Crystal Structure ◽  
Streptococcus Pneumoniae ◽  
Host Cells ◽  
Terminal Domain ◽  
Loop Region ◽  
C Terminus ◽  
Fibronectin Binding Protein ◽  
Fibronectin Binding ◽  
Α Helix ◽  
Domain I

The Gram-positive bacterium Streptococcus pneumoniae, a major human pathogen, is a regular colonizer of the upper and lower respiratory tracts. Pneumococcal adherence and virulence factor A (PavA), a fibronectin-binding bacterial protein, from S. pneumoniae is an important facilitator of its colonization of host cells. In this study, the crystal structure of the N-terminal domain of PavA (SpPavA-N) determined at a resolution of 2.39 Å is reported. Each monomer of the dimeric protein consists of two domains (domains I and II) and a short α-helix (α6) at the C-terminus that are connected by elongated loops. Comparison of the SpPavA-N structure with that of its homolog from Streptococcus suis (FBPS-N) revealed differences in α5, α6 and the domain II/α6 inter-loop region within domain II. The α5 helix of FBPS-N folds back toward domain I, whereas in SpPavA-N it adopts an elongated rod shape.

scholarly journals Crystal structure of XCC3289 from Xanthomonas campestris: homology with the N-terminal substrate-binding domain of Lon peptidase

2020 ◽  
Vol 76 (10) ◽  
pp. 488-494
Author(s):  
Rahul Singh ◽  
Sonali Deshmukh ◽  
Ashwani Kumar ◽  
Venuka Durani Goyal ◽  
Ravindra D. Makde
Keyword(s):  
Crystal Structure ◽  
Protein Interactions ◽  
Xanthomonas Campestris ◽  
Protein Quality ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
Homologous Proteins ◽  
Terminal Domain ◽  
Α Helix ◽  
Terminal Domains

LonA peptidase is a major component of the protein quality-control mechanism in both prokaryotes and the organelles of eukaryotes. Proteins homologous to the N-terminal domain of LonA peptidase, but lacking its other domains, are conserved in several phyla of prokaryotes, including the Xanthomonadales order. However, the function of these homologous proteins (LonNTD-like proteins) is not known. Here, the crystal structure of the LonNTD-like protein from Xanthomonas campestris (XCC3289; UniProt Q8P5P7) is reported at 2.8 Å resolution. The structure was solved by molecular replacement and contains one polypeptide in the asymmetric unit. The structure was refined to an R free of 29%. The structure of XCC3289 consists of two domains joined by a long loop. The N-terminal domain (residues 1–112) consists of an α-helix surrounded by β-sheets, whereas the C-terminal domain (residues 123–193) is an α-helical bundle. The fold and spatial orientation of the two domains closely resembles those of the N-terminal domains of the LonA peptidases from Escherichia coli and Mycobacterium avium. The structure is also similar to that of cereblon, a substrate-recognizing component of the E3 ubiquitin ligase complex. The N-terminal domains of both LonA and cereblon are known to be involved in specific protein–protein interactions. This structural analysis suggests that XCC3289 and other LonNTD-like proteins might also be capable of such protein–protein interactions.

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