首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 0 毫秒
1.
Recently, Zika virus (ZIKV) emerged as a global public health concern and is distinct from other flaviviruses in many aspects, for example, causing transplacental infection, fetal abnormalities and vector-independent transmission through body fluids in humans. The capsid (C) protein is a multifunctional protein, since it binds to viral RNA in the process of nucleocapsid assembly and plays important roles in virus infection processes by interacting with cellular proteins, modulating cellular metabolism, apoptosis and immune response. Here we solved the crystal structure of ZIKV C protein at a resolution of 1.9 Å. The ZIKV C protein structure contains four α helices with a long pre-α1 loop and forms dimers. The unique long pre-α1 loop in ZIKV C contributes to the tighter association of dimeric assembly and renders a divergent hydrophobic feature at the lipid bilayer interface in comparison with the known C structures of West Nile and dengue viruses. We reported the interaction between the ZIKV C protein and lipid droplets through confocal microscopy analysis. Substitutions of key amino acids in the pre-α1 loop of ZIKV C disrupted the interaction with lipid droplets, indicating that the loop is critical for membrane association. We also recognized that ZIKV C protein possesses broad binding capability to different nucleotide types, including single-stranded and double-stranded RNAs or DNAs. Furthermore, the highly positively charged interface, mainly formed by α4 helix, is proposed to be responsible for nucleotide binding. These findings will greatly enhance our understanding of ZIKV C protein, providing information for anti-ZIKV drug design targeting the C protein.  相似文献   

2.
TonB is a key protein in active transport of essential nutrients like vitamin B12 and metal sources through the outer membrane transporters of Gram-negative bacteria. This inner membrane protein spans the periplasm, contacts the outer membrane receptor by its periplasmic domain and transduces energy from the cytoplasmic membrane pmf to the receptor allowing nutrient internalization. Whereas generally a single TonB protein allows the acquisition of several nutrients through their cognate receptor, in some species one particular TonB is dedicated to a specific system. Despite a considerable amount of data available, the molecular mechanism of TonB-dependent active transport is still poorly understood. In this work, we present a structural study of a TonB-like protein, HasB dedicated to the HasR receptor. HasR acquires heme either free or via an extracellular heme transporter, the hemophore HasA. Heme is used as an iron source by bacteria. We have solved the structure of the HasB periplasmic domain of Serratia marcescens and describe its interaction with a critical region of HasR. Some important differences are observed between HasB and TonB structures. The HasB fold reveals a new structural class of TonB-like proteins. Furthermore, we have identified the structural features that explain the functional specificity of HasB. These results give a new insight into the molecular mechanism of nutrient active transport through the bacterial outer membrane and present the first detailed structural study of a specific TonB-like protein and its interaction with the receptor.  相似文献   

3.
Flavivirus RNA replication occurs within a replication complex (RC) that assembles on ER membranes and comprises both non-structural (NS) viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent-RNA polymerase (RdRp) domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3) at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV), the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5.  相似文献   

4.
Lantibiotics are peptide-derived antibiotics that inhibit the growth of Gram-positive bacteria via interactions with lipid II and lipid II-dependent pore formation in the bacterial membrane. Due to their general mode of action the Gram-positive producer strains need to express immunity proteins (LanI proteins) for protection against their own lantibiotics. Little is known about the immunity mechanism protecting the producer strain against its own lantibiotic on the molecular level. So far, no structures have been reported for any LanI protein. We solved the structure of SpaI, a LanI protein from the subtilin producing strain Bacillus subtilis ATCC 6633. SpaI is a 16.8-kDa lipoprotein that is attached to the outside of the cytoplasmic membrane via a covalent diacylglycerol anchor. SpaI together with the ABC transporter SpaFEG protects the B. subtilis membrane from subtilin insertion. The solution-NMR structure of a 15-kDa biologically active C-terminal fragment reveals a novel fold. We also demonstrate that the first 20 N-terminal amino acids not present in this C-terminal fragment are unstructured in solution and are required for interactions with lipid membranes. Additionally, growth tests reveal that these 20 N-terminal residues are important for the immunity mediated by SpaI but most likely are not part of a possible subtilin binding site. Our findings are the first step on the way of understanding the immunity mechanism of B. subtilis in particular and of other lantibiotic producing strains in general.  相似文献   

5.
6.
UL21 is a conserved protein in the tegument of alphaherpesviruses and has multiple important albeit poorly understood functions in viral replication and pathogenesis. To provide a roadmap for exploration of the multiple roles of UL21, we determined the crystal structure of its conserved N-terminal domain from herpes simplex virus 1 to 2.0-Å resolution, which revealed a novel sail-like protein fold. Evolutionarily conserved surface patches highlight residues of potential importance for future targeting by mutagenesis.  相似文献   

7.

Background

The replication of coronaviruses takes place on cytoplasmic double membrane vesicles (DMVs) originating in the endoplasmic reticulum (ER). Three trans-membrane non-structural proteins, nsp3, nsp4 and nsp6, are understood to be membrane anchors of the coronavirus replication complex. Nsp4 is localized to the ER membrane when expressed alone but is recruited into the replication complex in infected cells. It is revealed to contain four trans-membrane regions and its N- and C-termini are exposed to the cytosol.

Methodology/Principal Findings

We have determined the crystal structures of the C-terminal hydrophilic domain of nsp4 (nsp4C) from MHV strain A59 and a C425S site-directed mutant. The highly conserved 89 amino acid region from T408 to Q496 is shown to possess a new fold. The wild-type (WT) structure features two monomers linked by a Cys425-Cys425 disulfide bond in one asymmetric unit. The monomers are arranged with their N- and C-termini in opposite orientations to form an “open” conformation. Mutation of Cys425 to Ser did not affect the monomer structure, although the mutant dimer adopts strikingly different conformations by crystal packing, with the cross-linked C-termini and parallel N-termini of two monomers forming a “closed” conformation. The WT nsp4C exists as a dimer in solution and can dissociate easily into monomers in a reducing environment.

Conclusions/Significance

As nsp4C is exposed in the reducing cytosol, the monomer of nsp4C should be physiological. This structure may serve as a basis for further functional studies of nsp4.  相似文献   

8.
9.
10.
《Journal of molecular biology》2019,431(17):3179-3190
Stress proteins promote cell survival by monitoring protein homeostasis in cells and organelles. YcjX is a conserved protein of unknown function, which is highly upregulated in response to acute and chronic stress. Notably, heat shock induction of ycjX exceeded even levels observed for major stress-induced chaperones, including GroEL, ClpB, and HtpG, which use ATP as energy source. YcjX features a Walker-type nucleotide-binding domain indicating that YcjX might function as a molecular chaperone. Here, we present the first crystal structure of YcjX from Shewanella oneidensis solved at 1.9-Å resolution by SAD phasing. We show that YcjX is a GTP-binding protein that shares at its core the canonical alpha-beta domain of p21ras (Ras). However, unlike Ras, YcjX features several unique insertions, including an entirely α-helical domain not previously observed in Ras-like GTPases. We note that this helical domain is reminiscent of a similar domain in the Gα subunit of heterotrimeric G proteins, supporting a potential role for YcjX as a signal transducer of stress responses. To elucidate the mechanism of GTP hydrolysis, we determined crystal structures of YcjX bound to GDP and GDPCP, respectively, which crystallized in three different nucleotide switch conformations. Supported by targeted mutagenesis experiments, we show that YcjX utilizes a non-canonical switch 2′ motif not previously observed in Ras-like GTPases. Together, our structures provide atomic snapshots of YcjX in different functional states, illustrating the structural determinants for stress signaling.  相似文献   

11.
12.
In Bacillus subtilis, LytE, LytF, CwlS, and CwlO are vegetative autolysins, dl-endopeptidases in the NlpC/P60 family, and play essential roles in cell growth and separation. IseA (YoeB) is a proteinaceous inhibitor against the dl-endopeptidases, peptidoglycan hydrolases. Overexpression of IseA caused significantly long chained cell morphology, because IseA inhibits the cell separation dl-endopeptidases post-translationally. Here, we report the first three-dimensional structure of IseA, determined by NMR spectroscopy. The structure includes a single domain consisting of three α-helices, one 310-helix, and eight β-strands, which is a novel fold like a “hacksaw.” Noteworthy is a dynamic loop between β4 and the 310-helix, which resembles a “blade.” The electrostatic potential distribution shows that most of the surface is positively charged, but the region around the loop is negatively charged. In contrast, the LytF active-site cleft is expected to be positively charged. NMR chemical shift perturbation of IseA interacting with LytF indicated that potential interaction sites are located around the loop. Furthermore, the IseA mutants D100K/D102K and G99P/G101P at the loop showed dramatic loss of inhibition activity against LytF, compared with wild-type IseA, indicating that the β4–310 loop plays an important role in inhibition. Moreover, we built a complex structure model of IseA-LytF by docking simulation, suggesting that the β4–310 loop of IseA gets stuck deep in the cleft of LytF, and the active site is occluded. These results suggest a novel inhibition mechanism of the hacksaw-like structure, which is different from known inhibitor proteins, through interactions around the characteristic loop regions with the active-site cleft of enzymes.  相似文献   

13.
14.
The novel tumor biomarker MIEN1, identified by representational difference analysis, is overexpressed in breast cancer and prostate cancer. MIEN1 is considered an oncogenic protein, because MIEN1 overexpression functionally enhances migration and invasion of tumor cells via modulating the activity of AKT. However, the structure and molecular function of MIEN1 is little understood. Here, we report the solution structure of MIEN1, which adopts a thioredoxin-like fold with a redox-active motif. Comparison of backbone chemical shifts showed that most of the residues for both oxidized and reduced MIEN1 possessed the same backbone conformation, with differences limited to the active motif and regions in proximity. The redox potential of this disulfide bond was measured as −225 mV, which compares well with that of disulfides for other thioredoxin-like proteins. Overall, our results suggest that MIEN1 may have an important regulatory role in phosphorylation of AKT with its redox potential.  相似文献   

15.
Lactococcus lactis, a gram-positive bacterium widely used by the dairy industry to manufacture cheeses, is subject to infection by a diverse population of virulent phages. We have previously determined the structures of three receptor binding proteins (RBPs) from lactococcal phages TP901-1, p2, and bIL170, each of them having a distinct host range. Virulent phages p2 and bIL170 are classified within the 936 group, while the temperate phage TP901-1 is a member of the genetically distinct P335 polythetic group. These RBPs comprise three domains: the N-terminal domain, binding to the virion particle; a β-helical linker domain; and the C-terminal domain, bearing the receptor binding site used for host recognition. Here, we have designed, expressed, and determined the structure of an RBP chimera in which the N-terminal and linker RBP domains of phage TP901-1 (P335) are fused to the C-terminal RBP domain of phage p2 (936). This chimera exhibits a stable structure that closely resembles the parental structures, while a slight displacement of the linker made RBP domain adaptation efficient. The receptor binding site is structurally indistinguishable from that of native p2 RBP and binds glycerol with excellent affinity.A broad number of products are manufactured by large-scale bacterial fermentation, including the value-added fermented dairy products. Most bacterial fermentation industries have experienced problems with phage contamination. Phage outbreaks are costly and time-consuming because they can slow or arrest the fermentation process and adversely affect product quality (15). For decades, the dairy industry has relied on an array of strategies to control this natural phenomenon, including rotation of their bacterial cultures (11, 24, 25). However, in spite of these efforts, new virulent lactococcal phages keep emerging. A better understanding of the various mechanisms affecting the genetic diversity of the phage population is necessary for optimal phage control strategies (18).Lactococcal phages are among the most studied bacterial viruses because of the economic importance of their hosts. Hundreds of lactococcal phages have been isolated, and the vast majority of them have a long, contractile tail, thereby belonging to the Siphoviridae family (1). Lactococcus lactis phages are currently classified into 10 genetically distinct groups (10), but only members of 3 of them are highly adapted to multiply in milk, namely, the 936, c2, and P335 groups (11, 24, 25). The first step for such an effective viral infection is host recognition, which necessitates the interaction between the adsorption device located at the distal tail end of the phage and the cell surface receptor (32). Members of the 936 and P335 groups recognize their host through an interaction between their receptor binding protein (RBP) (13) and receptors, probably lipoteichoic acids, at the host cell surface (27, 29-31).We have previously determined the crystal structures of three RBPs, from the virulent lactococcal phages p2 (30, 31) and bIL170 (936 group) (27) and from the temperate phage TP901-1 (P335 group) (29). The RBPs of these phages have a similar architecture of three protomers related by a threefold axis. Each protomer comprises three domains: the N terminus (named shoulders in p2), the interlaced β-prism linker (the “neck” domain), and the jelly-roll domain (2) at the C terminus (the “head” domain). This last domain harbors a saccharide binding site likely involved in host recognition, as it binds with high affinity to phosphoglycerol, a component of teichoic acid (8, 19, 27, 29-31). We have previously shown that the shoulder and neck domains are highly conserved in the RBPs of 936-like phages (8, 19, 27, 29-31). The individuality of the RBP C-terminal domain sequence likely dictates phage specificity for the receptor, which may specifically recognize different substitutions (H, GlcNAc, or d-Ala) of the phosphoglycerol moieties of the L. lactis teichoic acid polymers. Recently, the complete genomic sequence of the reference virulent phage P335 was determined, and comparative analysis revealed that the C terminus of its RBP showed homology to the RBP of the virulent lactococcal phage P475 of the 936 group (17). Such homology between RBP head domains was surprising because the two lactococcal phage groups rarely shared common genes or domains. This observation suggested that modular shuffling of domains can occur between these otherwise genetically distinct phage groups.The overall fold of the N-terminal RBP domain is different in 936- and P335-like phages. In the P335 group, the N-terminal domain comprises a unique helix that fits into the rest of the phage baseplate (28, 29) (Fig. (Fig.1A),1A), while in the 936 group, this 140-residue domain is a large β-sandwich with an external α-helix (30) (Fig. (Fig.1B).1B). Nonetheless, the N-terminal domains of the two RBPs may still be, related because both appear to be built using a coiled coil, although the 936-like phages have an additional β-sandwich. The β-prism linkers (neck domain) of the two phage groups also differ in sequence and in radius, but they have a similar fold, the latter being also close to that of T4 phage short fiber (33). The linker domain of phage TP901-1 is wider than that of p2 and exhibits a repeated motif (G-X-Y-X-Y, where X is polar and Y nonpolar). Finally, the C-terminal domains of both species share the same fold, a jelly-roll motif (2) also found in adenovirus (5) and reovirus (3, 4, 6).Open in a separate windowFIG. 1.Structures and sequences of RBPs from lactococcal phages. (A) Three-dimensional structure of the RBP from phage TP901-1 (P335 group; blue). (B) Three-dimensional structure of the RBP from phage p2 (936 group; magenta). (C) View of a model associating domains of TP901-1 (N terminus and linker domain, below red line, blue) and p2 (head, above red line, magenta) RBPs. (D) Three-dimensional crystal structure of chimera form 1 (yellow) assembled according to the model in panel C. (E) Sequence alignment of the RBPs of p2 (part) and TP901-1. The secondary structure is described above the alignment. The binding residues are shown with blue dots. The hinge proline (Pro 162/63) is identified by a red arrow. The chimera is composed of the N-terminal domain (residues 17 to 33) and the linker domain residues (residues 34 to 63) from phage TP901-1 RBP and the C-terminal domain (residues 163 to 264) from phage p2 RBP.The question addressed here was whether exchange between the C-terminal domains of two phage groups would lead to a stable protein with conserved binding capacity. To answer this question, we have generated an RBP chimera comprising the N-terminal and linker domains of phage TP901-1 fused to the C-terminal domain of phage p2. We have produced this chimera and determined its crystal structure and its sugar binding capacity. These results indicate that straightforward domain exchange produced a stable chimera with a conserved binding capacity and a structure close to that of each of the parental parts.  相似文献   

16.
17.
CCM3 mutations are associated with cerebral cavernous malformation (CCM), a disease affecting 0.1–0.5% of the human population. CCM3 (PDCD10, TFAR15) is thought to form a CCM complex with CCM1 and CCM2; however, the molecular basis for these interactions is not known. We have determined the 2.5 Å crystal structure of CCM3. This structure shows an all α-helical protein containing two domains, an N-terminal dimerization domain with a fold not previously observed, and a C-terminal focal adhesion targeting (FAT)-homology domain. We show that CCM3 binds CCM2 via this FAT-homology domain and that mutation of a highly conserved FAK-like hydrophobic pocket (HP1) abrogates CCM3-CCM2 interaction. This CCM3 FAT-homology domain also interacts with paxillin LD motifs using the same surface, and partial CCM3 co-localization with paxillin in cells is lost on HP1 mutation. Disease-related CCM3 truncations affect the FAT-homology domain suggesting a role for the FAT-homology domain in the etiology of CCM.  相似文献   

18.
  1. Download : Download high-res image (417KB)
  2. Download : Download full-size image
  相似文献   

19.
Phanta is a reversibly photoswitching chromoprotein (ΦF, 0.003), useful for pcFRET, that was isolated from a mutagenesis screen of the bright green fluorescent eCGP123 (ΦF, 0.8). We have investigated the contribution of substitutions at positions His193, Thr69 and Gln62, individually and in combination, to the optical properties of Phanta. Single amino acid substitutions at position 193 resulted in proteins with very low ΦF, indicating the importance of this position in controlling the fluorescence efficiency of the variant proteins. The substitution Thr69Val in Phanta was important for supressing the formation of a protonated chromophore species observed in some His193 substituted variants, whereas the substitution Gln62Met did not significantly contribute to the useful optical properties of Phanta. X-ray crystal structures for Phanta (2.3 Å), eCGP123T69V (2.0 Å) and eCGP123H193Q (2.2 Å) in their non-photoswitched state were determined, revealing the presence of a cis-coplanar chromophore. We conclude that changes in the hydrogen-bonding network supporting the cis-chromophore, and its contacts with the surrounding protein matrix, are responsible for the low fluorescence emission of eCGP123 variants containing a His193 substitution.  相似文献   

20.
Crimean-Congo hemorrhagic fever virus (CCHFV) is an emerging tick-borne virus of the Bunyaviridae family that is responsible for a fatal human disease for which preventative or therapeutic measures do not exist. We solved the crystal structure of the CCHFV strain Baghdad-12 nucleocapsid protein (N), a potential therapeutic target, at a resolution of 2.1 Å. N comprises a large globular domain composed of both N- and C-terminal sequences, likely involved in RNA binding, and a protruding arm domain with a conserved DEVD caspase-3 cleavage site at its apex. Alignment of our structure with that of the recently reported N protein from strain YL04057 shows a close correspondence of all folds but significant transposition of the arm through a rotation of 180 degrees and a translation of 40 Å. These observations suggest a structural flexibility that may provide the basis for switching between alternative N protein conformations during important functions such as RNA binding and oligomerization. Our structure reveals surfaces likely involved in RNA binding and oligomerization, and functionally critical residues within these domains were identified using a minigenome system able to recapitulate CCHFV-specific RNA synthesis in cells. Caspase-3 cleaves the polypeptide chain at the exposed DEVD motif; however, the cleaved N protein remains an intact unit, likely due to the intimate association of N- and C-terminal fragments in the globular domain. Structural alignment with existing N proteins reveals that the closest CCHFV relative is not another bunyavirus but the arenavirus Lassa virus instead, suggesting that current segmented negative-strand RNA virus taxonomy may need revision.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号