Functional characterization and localization of the TcpH conjugation protein from Clostridium perfringens

In Clostridium perfringens, conjugative plasmids encode important virulence factors, such as toxins and resistance determinants. All of these plasmids carry a conjugation locus that consists of 11 genes: intP and tcpA to tcpJ. Three proteins, TcpA, a potential coupling protein, TcpF, a putative ATPase that is similar to ORF15 from Tn916, and TcpH, which contains VirB6-like domains, are essential for conjugation in the prototype conjugative plasmid pCW3. To analyze the functional domains of TcpH, a putative structural component of the mating-pair formation complex and deletion and site-directed mutants were constructed and analyzed. The results showed that the N-terminal 581 residues and the conserved (242)VQQPW(246) motif were required for conjugative transfer. Bacterial two-hybrid and biochemical studies showed that TcpH interacted with itself and with TcpC. An analysis of the tcpH mutants demonstrated that the region required for these interactions also was localized to the N-terminal 581 residues and that the function of the C-terminal region of TcpH was independent of protein-protein interactions. Finally, immunofluorescence studies showed that TcpH and TcpF were located at both cell poles of donor C. perfringens cells. The results provide evidence that TcpH is located in the cell membrane, where it oligomerizes and interacts with TcpC to form part of the mating-pair formation complex, which is located at the cell poles and is closely associated with TcpF.     

Disruption of LptA oligomerization and affinity of the LptA–LptC interaction

The lipopolysaccharide (LPS)‐rich outer membrane (OM) is a unique feature of Gram‐negative bacteria, and LPS transport across the inner membrane (IM) and through the periplasm is essential to the biogenesis and maintenance of the OM. LPS is transported across the periplasm to the outer leaflet of the OM by the LPS transport (Lpt) system, which in Escherichia coli is comprised of seven recently identified proteins, including LptA, LptC, LptDE, and LptFGB₂. Structures of the periplasmic protein LptA and the soluble portion of the membrane‐associated protein LptC have been solved and show these two proteins to be highly structurally homologous with unique folds. LptA has been shown to form concentration dependent oligomers that stack end‐to‐end. LptA and LptC have been shown to associate in vivo and are expected to form a similar protein–protein interface to that found in the LptA dimer. In these studies, we disrupted LptA oligomerization by introducing two point mutations that removed a lysine and glutamine side chain from the C‐terminal β‐strand of LptA. This loss of oligomerization was characterized using EPR spectroscopy techniques and the affinity of the interaction between the mutant LptA protein and WT LptC was determined using EPR spectroscopy (K_d = 15 µM) and isothermal titration calorimetry (K_d = 14 µM). K_d values were also measured by EPR spectroscopy for the interaction between LptC and WT LptA (4 µM) and for WT LptA oligomerization (29 µM). These data suggest that the affinity between LptA and LptC is stronger than the affinity for LptA oligomerization.     

Crystal structure of the ubiquitin‐like small archaeal modifier protein 2 from Haloferax volcanii

The discovery of ubiquitin‐like small archaeal modifier protein 2 (SAMP2) that forms covalent polymeric chains in Haloferax volcanii has generated tremendous interest in the function and regulation of this protein. At present, it remains unclear whether the Hfx. volcanii modifier protein SAMP1 has such polyubiquitinating‐like activity. Although SAMP1 and SAMP2 use the same conjugation machinery to modify their target proteins, each can impart distinct functional consequences. To better understand the mechanism of SAMP2 conjugation, we have sought to characterize the biophysical and structural properties of the protein from Hfx. volcanii. SAMP2 is only partially structured under mesohalic solution conditions and adopts a well‐folded compact conformation in the presence of 2.5M of NaCl. Its 2.3‐Å‐resolution crystal structure reveals a characteristic α/β central core domain and a unique β‐hinge motif. This motif anchors an unusual C‐terminal extension comprising the diglycine tail as well as two lysine residues that can potentially serve to interlink SAMP2 moieties. Mutational alternation of the structural malleability of this β‐hinge motif essentially abolishes the conjugation activity of SAMP2 in vivo. In addition, NMR structural studies of the putative ubiquitin‐like protein HVO_2177 from Hfx. volcanii show that like SAMP1, HVO_2177 forms a classic β‐grasp fold in a salt‐independent manner. These results provide insights into the structure–function relationship of sampylating proteins of fundamental importance in post‐translational protein modification and environmental cues in Archaea.     

The N-terminal amphipathic region of the Escherichia coli type III secretion system protein EspD is required for membrane insertion and function

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. These pathogenic E. coli express a syringe‐like protein machine, known as the type III secretion system (T3SS), used for the injection of virulence factors into the cytosol of the host epithelial cell. Breaching the epithelial plasma membrane requires formation of a translocation pore that contains the secreted protein EspD. Here we demonstrate that the N‐terminal segment of EspD, encompassing residues 1–171, contains two amphipathic domains spanning residues 24–41 and 66–83, with the latter of these helices being critical for EspD function. Fluorescence and circular dichroism analysis revealed that, in solution, His₆‐EspD_1–171 adopts a native disordered structure; however, on binding anionic small unilamellar vesicles composed of phosphatidylserine, His₆‐EspD_1–171 undergoes a pH depended conformational change that increases the α‐helix content of this protein approximately sevenfold. This change coincides with insertion of the region circumscribing Trp₄₇ into the hydrophobic core of the lipid bilayer. On the HeLa cell plasma membrane, His₆‐EspD_1–171 forms a homodimer that is postulated to promote EspD–EspD oligomerization and pore formation. Complementation of ΔespD null mutant bacteria with an espDΔ66–83 gene showed that this protein was secreted but non‐functional.     

Crystal structure of the Haemophilus influenzae Hap adhesin reveals an intercellular oligomerization mechanism for bacterial aggregation

Bacterial biofilms are complex microbial communities that are common in nature and are being recognized increasingly as an important determinant of bacterial virulence. However, the structural determinants of bacterial aggregation and eventual biofilm formation have been poorly defined. In Gram‐negative bacteria, a major subgroup of extracellular proteins called self‐associating autotransporters (SAATs) can mediate cell–cell adhesion and facilitate biofilm formation. In this study, we used the Haemophilus influenzae Hap autotransporter as a prototype SAAT to understand how bacteria associate with each other. The crystal structure of the H. influenzae Hap_S passenger domain (harbouring the SAAT domain) was determined to 2.2 Å by X‐ray crystallography, revealing an unprecedented intercellular oligomerization mechanism for cell–cell interaction. The C‐terminal SAAT domain folds into a triangular‐prism‐like structure that can mediate Hap–Hap dimerization and higher degrees of multimerization through its F1–F2 edge and F2 face. The intercellular multimerization can give rise to massive buried surfaces that are required for overcoming the repulsive force between cells, leading to bacterial cell–cell interaction and formation of complex microcolonies.     

Dimerization of a flocculent protein from Moringa oleifera: experimental evidence and in silico interpretation

Many proteins exist in dimeric and other oligomeric forms to gain stability and functional advantages. In this study, the dimerization property of a coagulant protein (MO_2.1) from Moringa oleifera seeds was addressed through laboratory experiments, protein–protein docking studies and binding free energy calculations. The structure of MO_2.1 was predicted by homology modelling, while binding free energy and residues-distance profile analyses provided insight into the energetics and structural factors for dimer formation. Since the coagulation activities of the monomeric and dimeric forms of MO_2.1 were comparable, it was concluded that oligomerization does not affect the biological activity of the protein.     

Noncollagenous region of the streptococcal collagen‐like protein is a trimerization domain that supports refolding of adjacent homologous and heterologous collagenous domains

Proper folding of the (Gly‐Xaa‐Yaa)_n sequence of animal collagens requires adjacent N‐ or C‐terminal noncollagenous trimerization domains which often contain coiled‐coil or beta sheet structure. Collagen‐like proteins have been found recently in a number of bacteria, but little is known about their folding mechanism. The Scl2 collagen‐like protein from Streptococcus pyogenes has an N‐terminal globular domain, designated V_sp, adjacent to its triple‐helix domain. The V_sp domain is required for proper refolding of the Scl2 protein in vitro. Here, recombinant V_sp domain alone is shown to form trimers with a significant α‐helix content and to have a thermal stability of T_m = 45°C. Examination of a new construct shows that the V_sp domain facilitates efficient in vitro refolding only when it is located N‐terminal to the triple‐helix domain but not when C‐terminal to the triple‐helix domain. Fusion of the V_sp domain N‐terminal to a heterologous (Gly‐Xaa‐Yaa)_n sequence from Clostridium perfringens led to correct folding and refolding of this triple‐helix, which was unable to fold into a triple‐helical, soluble protein on its own. These results suggest that placement of a functional trimerization module adjacent to a heterologous Gly‐Xaa‐Yaa repeating sequence can lead to proper folding in some cases but also shows specificity in the relative location of the trimerization and triple‐helix domains. This information about their modular nature can be used in the production of novel types of bacterial collagen for biomaterial applications.     

The Putative Coupling Protein TcpA Interacts with Other pCW3-Encoded Proteins To Form an Essential Part of the Conjugation Complex

Conjugative plasmids encode antibiotic resistance determinants or toxin genes in the anaerobic pathogen Clostridium perfringens. The paradigm conjugative plasmid in this bacterium is pCW3, a 47-kb tetracycline resistance plasmid that encodes the unique tcp transfer locus. The tcp locus consists of 11 genes, intP and tcpA-tcpJ, at least three of which, tcpA, tcpF, and tcpH, are essential for the conjugative transfer of pCW3. In this study we examined protein-protein interactions involving TcpA, the putative coupling protein. Use of a bacterial two-hybrid system identified interactions between TcpA and TcpC, TcpG, and TcpH. This analysis also demonstrated TcpA, TcpC, and TcpG self-interactions, which were confirmed by chemical cross-linking studies. Examination of a series of deletion and site-directed derivatives of TcpA identified the domains and motifs required for these interactions. Based on these results, we have constructed a model for this unique conjugative transfer apparatus.Conjugation systems are important contributors to the dissemination of antibiotic resistance determinants and virulence factors. Extensive analysis of conjugative plasmids from gram-negative bacteria has led to the elucidation of a general mechanism of conjugative transfer (10, 22). In this process, the transferred DNA is processed by components of a relaxosome complex. Specifically, the DNA is nicked at the origin of transfer (oriT) by a relaxase, which remains covalently coupled to the transferred DNA strand. The single-stranded DNA complex then interacts with the coupling protein, a DNA-dependent ATPase that provides the energy to actively pump the DNA through the mating pair formation (Mpf) complex into the recipient cell (36). The coupling protein interacts with both DNA processing proteins and components of the Mpf complex (1, 4, 12, 35, 38). These interactions have been demonstrated using bacterial and yeast two-hybrid approaches as well as gel filtration, pull-down, and coimmunoprecipitation studies.The mechanism of conjugative transfer has yet to be precisely determined for conjugative plasmids from gram-positive bacteria although bioinformatics analysis has identified similar gene arrangements and conservation of gene sequences within the transfer regions encoded on conjugative plasmids identified from strains of streptococcal, staphylococcal, enterococcal, and lactococcal origin (15). It was proposed that gram-positive and gram-negative conjugation systems utilize a similar transfer mechanism (15).In the anaerobic pathogen Clostridium perfringens conjugative plasmids have been shown to encode antibiotic resistance genes or extracellular toxins (3, 8, 9, 18). Although the contribution of conjugation to disease dissemination has not been systematically evaluated, it has been proposed that transfer of the C. perfringens enterotoxin plasmid pCPF4969 to normal flora isolates of C. perfringens may contribute to the severity of disease caused by non-food-borne isolates of C. perfringens (9).The prototype conjugative plasmid in C. perfringens is the 47-kb tetracycline resistance plasmid, pCW3. The complete sequence of pCW3 has been determined, and its unique replication protein and conjugation locus have been identified (8). Bioinformatics analysis of this C. perfringens tcp conjugation locus identified several proteins with limited similarity to proteins encoded within the transfer region of the conjugative transposon, Tn916 (8). The role of the tcp locus in the transfer of pCW3 has been confirmed by isolation of independent tcpA, tcpF, and tcpH mutants and subsequent complementation studies (8, 29). Since the region that encompasses the tcp locus is conserved in all conjugative plasmids from C. perfringens (2, 3, 8, 9, 18, 27) and since divergent tcpA homologues can complement a pCW3tcpA mutant (29), it appears that the conjugative transfer of both antibiotic resistance and toxin plasmids from this bacterium utilizes a common but poorly understood mechanism. Note that the C. perfringens tcp conjugation locus is different from the transfer regions of conjugative plasmids from other gram-positive bacteria.We have recently shown that the essential conjugation protein TcpH, a putative membrane-associated Mpf complex component, is localized to the poles of C. perfringens cells, as is another essential conjugation protein, TcpF (37). TcpH has also been shown to interact with itself and with the pCW3-encoded TcpC protein (37). In this study we have focused on the essential conjugation protein TcpA. Since TcpA encodes an FtsK/SpoIIIE domain found in DNA translocases (8), it is proposed that TcpA is involved in the movement of DNA during conjugative transfer, fulfilling a role equivalent to that of coupling proteins in other conjugation systems. Like such proteins, TcpA encodes two N-terminal transmembrane domains (TMDs) and a C-terminal cytoplasmic region that contains three motifs predicted to be involved in ATP binding and hydrolysis (8). Our previous studies revealed that the conserved motifs, motif I (Walker A box), motif II (Walker B box), and motif III (RAAG box), are essential for the function of TcpA. The C-terminal 61 amino acids (aa), though not essential for TcpA function, were shown to be important for efficient transfer of pCW3, as were the putative TMDs (29).To further investigate pCW3 transfer and the role of TcpA in this process, we have used bacterial two-hybrid analysis to examine protein-protein interactions involving TcpA. Using this system, interactions were observed between TcpA and itself, TcpC, TcpG, and TcpH. In addition, TcpC and TcpG were also found to self-interact. By combining these data with other data generated in this laboratory (37), we have constructed a model for the conjugative transfer of pCW3.     

TcpM: a novel relaxase that mediates transfer of large conjugative plasmids from Clostridium perfringens

Conjugative transfer of toxin and antibiotic resistance plasmids in Clostridium perfringens is mediated by the tcp conjugation locus. Surprisingly, neither a relaxase gene nor an origin of transfer (oriT) has been identified on these plasmids, which are typified by the 47 kb tetracycline resistance plasmid pCW3. The tcpM gene (previously called intP) encodes a potential tyrosine recombinase that was postulated to be an atypical relaxase. Mutagenesis and complementation studies showed that TcpM was required for wild‐type transfer of pCW3 and that a tyrosine residue, Y259, was essential for TcpM activity, which was consistent with the need for a relaxase‐mediated hydrophilic attack at the oriT site. Other catalytic residues conserved in tyrosine recombinases were not required for TcpM activity, suggesting that TcpM was not a site‐specific recombinase. Mobilization studies led to the identification of the oriT site, which was located in the 391 bp intergenic region upstream of tcpM. The oriT site was localized to a 150 bp region, and gel mobility shift studies showed that TcpM could bind to this region. Based on these studies we postulate that conjugative transfer of pCW3 involves the atypical relaxase TcpM binding to and processing the oriT site to initiate plasmid transfer.     

Vitronectin binds to the head region of Moraxella catarrhalis ubiquitous surface protein A2 and confers complement‐inhibitory activity

The serum resistance of the common respiratory pathogen Moraxella catarrhalis is mainly dependent on ubiquitous surface proteins (Usp) A1 and A2 that interact with complement factor 3 (C3) and complement inhibitor C4b binding protein (C4BP) preventing the alternative and classical pathways of the complement system respectively. UspA2 also has the capacity to attract vitronectin that in turn binds C9 and hereby inhibits membrane attack complex (MAC) formation. We found UspA2 as a major vitronectin binding protein and hence the UspA2/vitronectin interaction was studied in detail. The affinity constant (K_D) for vitronectin binding to UspA2 was 2.3 × 10^?8 M, and the N‐terminal region encompassing residues UspA2 30–170 bound vitronectin with a K_D of 7.9 × 10^?8 M. Electron microscopy verified that the active binding domain (UspA2^30–177) was located at the head region of UspA2. Experiments with recombinantly expressed vitronectin also revealed that UspA2^30–177 bound to the C‐terminal region of vitronectin residues 312–396. Finally, when human serum was pre‐incubated with UspA2, bacteria showed significantly less serum resistance. Our study directly reveals the binding mode between the N‐terminal domain of UspA2 and the C‐terminal part of vitronectin and thus sheds light upon the mechanism of M. catarrhalis‐dependent serum resistance.     

Development of a multifunctional tool for drug screening against plasmodial protein–protein interactions via surface plasmon resonance

We have developed an expression system capable of producing large quantities of low cost, specific peptides that are either His₁₂‐tagged, biotinylated, or unlabeled. The flexibility of this peptide system is suitable for interaction studies via surface plasmon resonance (SPR), co‐crystallization, and enzyme‐linked immunosorbent assay. Gene blocks containing peptide sequences of interest in addition to a 15 amino acid AviTag?, were cloned into a vector expressing an N‐terminal maltose binding protein. The constructs were expressed and purified, and the molecular weights of the recombinant proteins were estimated by analytical size exclusion chromatography. Successful in situ biotinylation of the AviTag was confirmed by anti‐biotin western blot and was used for coupling to the surface plasmon resonance chip. We were able to validate, as a proof of concept study, the specific protein–protein interaction of Plasmodium falciparum aldolase (PfAldolase) with three different cytoplasmic adhesin tail peptides from the family of thrombospondin‐related anonymous proteins (TRAPs), and to determine their affinities. This method of peptide production enables high yield production of peptides in a two‐day, cost effective manner. This tool will allow us to screen for protein–protein interaction inhibitors directed toward the liver stage and blood stage complexes of the glideosome in Plasmodium species. Adaptation of this tool will allow researchers to pursue their own studies of protein–protein interactions. Copyright © 2013 John Wiley & Sons, Ltd.     

Optimization of elastin‐like polypeptide fusions for expression and purification of recombinant proteins in plants

The demand for recombinant proteins for medical and industrial use is expanding rapidly and plants are now recognized as an efficient, inexpensive means of production. Although the accumulation of recombinant proteins in transgenic plants can be low, we have previously demonstrated that fusions with an elastin‐like polypeptide (ELP) tag can significantly enhance the production yield of a range of different recombinant proteins in plant leaves. ELPs are biopolymers with a repeating pentapeptide sequence (VGVPG)_n that are valuable for bioseparation, acting as thermally responsive tags for the non‐chromatographic purification of recombinant proteins. To determine the optimal ELP size for the accumulation of recombinant proteins and their subsequent purification, various ELP tags were fused to green fluorescent protein, interleukin‐10, erythropoietin and a single chain antibody fragment and then transiently expressed in tobacco leaves. Our results indicated that ELP tags with 30 pentapeptide repeats provided the best compromise between the positive effects of small ELP tags (n = 5–40) on recombinant protein accumulation and the beneficial effects of larger ELP tags (n = 80–160) on recombinant protein recovery during inverse transition cycling (ITC) purification. In addition, the C‐terminal orientation of ELP fusion tags produced higher levels of target proteins, relative to N‐terminal ELP fusions. Importantly, the ELP tags had no adverse effect on the receptor binding affinity of erythropoietin, demonstrating the inert nature of these tags. The use of ELP fusion tags provides an approach for enhancing the production of recombinant proteins in plants, while simultaneously assisting in their purification. Biotechnol. Bioeng. 2009;103: 562–573. © 2009 Wiley Periodicals, Inc.     

Oligomerization state and supramolecular structure of the HIV-1 Vpu protein transmembrane segment in phospholipid bilayers

HIV‐1 Vpu is an 81‐residue protein with a single N‐terminal transmembrane (TM) helical segment that is involved in the release of new virions from host cell membranes. Vpu and its TM segment form ion channels in phospholipid bilayers, presumably by oligomerization of TM helices into a pore‐like structure. We describe measurements that provide new constraints on the oligomerization state and supramolecular structure of residues 1–40 of Vpu (Vpu_1–40), including analytical ultracentrifugation measurements to investigate oligomerization in detergent micelles, photo‐induced crosslinking experiments to investigate oligomerization in bilayers, and solid‐state nuclear magnetic resonance measurements to obtain constraints on intermolecular contacts between and orientations of TM helices in bilayers. From these data, we develop molecular models for Vpu TM oligomers. The data indicate that a variety of oligomers coexist in phospholipid bilayers, so that a unique supramolecular structure can not be defined. Nonetheless, since oligomers of various sizes have similar intermolecular contacts and orientations, molecular models developed from our data are most likely representative of Vpu TM oligomers that exist in host cell membranes.     

Dynamic pH‐induced conformational changes of the PsbO protein in the fluctuating acidity of the thylakoid lumen

The PsbO protein is an essential extrinsic subunit of photosystem II, the pigment–protein complex responsible for light‐driven water splitting. Water oxidation in photosystem II supplies electrons to the photosynthetic electron transfer chain and is accompanied by proton release and oxygen evolution. While the electron transfer steps in this process are well defined and characterized, the driving forces acting on the liberated protons, their dynamics and their destiny are all largely unknown. It was suggested that PsbO undergoes proton‐induced conformational changes and forms hydrogen bond networks that ensure prompt proton removal from the catalytic site of water oxidation, i.e. the Mn₄CaO₅ cluster. This work reports the purification and characterization of heterologously expressed PsbO from green algae Chlamydomonas reinhardtii and two isoforms from the higher plant Solanum tuberosum (PsbO1 and PsbO2). A comparison to the spinach PsbO reveals striking similarities in intrinsic protein fluorescence and CD spectra, reflecting the near‐identical secondary structure of the proteins from algae and higher plants. Titration experiments using the hydrophobic fluorescence probe ANS revealed that eukaryotic PsbO proteins exhibit acid–base hysteresis. This hysteresis is a dynamic effect accompanied by changes in the accessibility of the protein's hydrophobic core and is not due to reversible oligomerization or unfolding of the PsbO protein. These results confirm the hypothesis that pH‐dependent dynamic behavior at physiological pH ranges is a common feature of PsbO proteins and causes reversible opening and closing of their β‐barrel domain in response to the fluctuating acidity of the thylakoid lumen.     

Crystallographic and mass spectrometric analyses of a tandem GNAT protein from the clavulanic acid biosynthesis pathway

(3R,5R)‐Clavulanic acid (CA) is a clinically important inhibitor of Class A β‐lactamases. Sequence comparisons suggest that orf14 of the clavulanic acid biosynthesis gene cluster encodes for an acetyl transferase (CBG). Crystallographic studies reveal CBG to be a member of the emerging structural subfamily of tandem Gcn5‐related acetyl transferase (GNAT) proteins. Two crystal forms (C2 and P2₁ space groups) of CBG were obtained; in both forms one molecule of acetyl‐CoA (AcCoA) was bound to the N‐terminal GNAT domain, with the C‐terminal domain being unoccupied by a ligand. Mass spectrometric analyzes on CBG demonstrate that, in addition to one strongly bound AcCoA molecule, a second acyl‐CoA molecule can bind to CBG. Succinyl‐CoA and myristoyl‐CoA displayed the strongest binding to the “second” CoA binding site, which is likely in the C‐terminal GNAT domain. Analysis of the CBG structures, together with those of other tandem GNAT proteins, suggest that the AcCoA in the N‐terminal GNAT domain plays a structural role whereas the C‐terminal domain is more likely to be directly involved in acetyl transfer. The available crystallographic and mass spectrometric evidence suggests that binding of the second acyl‐CoA occurs preferentially to monomeric rather than dimeric CBG. The N‐terminal AcCoA binding site and the proposed C‐terminal acyl‐CoA binding site of CBG are compared with acyl‐CoA binding sites of other tandem and single domain GNAT proteins. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Crystal structure of the protein At3g01520, a eukaryotic universal stress protein‐like protein from arabidopsis thaliana in complex with AMP

Members of the universal stress protein (USP) family are conserved in a phylogenetically diverse range of prokaryotes, fungi, protists, and plants and confer abilities to respond to a wide range of environmental stresses. Arabidopsis thaliana contains 44 USP domain‐containing proteins, and USP domain is found either in a small protein with unknown physiological function or in an N‐terminal portion of a multi‐domain protein, usually a protein kinase. Here, we report the first crystal structure of a eukaryotic USP‐like protein encoded from the gene At3g01520. The crystal structure of the protein At3g01520 was determined by the single‐wavelength anomalous dispersion method and refined to an R factor of 21.8% (R_free = 26.1%) at 2.5 Å resolution. The crystal structure includes three At3g01520 protein dimers with one AMP molecule bound to each protomer, comprising a Rossmann‐like α/β overall fold. The bound AMP and conservation of residues in the ATP‐binding loop suggest that the protein At3g01520 also belongs to the ATP‐binding USP subfamily members. Proteins 2015; 83:1368–1373. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

TcpA, an FtsK/SpoIIIE homolog, is essential for transfer of the conjugative plasmid pCW3 in Clostridium perfringens

The conjugative tetracycline resistance plasmid pCW3 is the paradigm conjugative plasmid in the anaerobic gram-positive pathogen Clostridium perfringens. Two closely related FtsK/SpoIIIE homologs, TcpA and TcpB, are encoded on pCW3, which is significant since FtsK domains are found in coupling proteins of gram-negative conjugation systems. To develop an understanding of the mechanism of conjugative transfer in C. perfringens, we determined the role of these proteins in the conjugation process. Mutation and complementation analysis was used to show that the tcpA gene was essential for the conjugative transfer of pCW3 and that the tcpB gene was not required for transfer. Furthermore, complementation of a pCW3DeltatcpA mutant with divergent tcpA homologs provided experimental evidence that all of the known conjugative plasmids from C. perfringens use a similar transfer mechanism. Functional genetic analysis of the TcpA protein established the essential role in conjugative transfer of its Walker A and Walker B ATP-binding motifs and its FtsK-like RAAG motif. It is postulated that TcpA is the essential DNA translocase or coupling protein encoded by pCW3 and as such represents a key component of the unique conjugation process in C. perfringens.     

Structural characterizations of the chloroplast translocon protein Tic110

Tic110 is a major component of the chloroplast protein import translocon. Two functions with mutually exclusive structures have been proposed for Tic110: a protein‐conducting channel with six transmembrane domains and a scaffold with two N‐terminal transmembrane domains followed by a large soluble domain for binding transit peptides and other stromal translocon components. To investigate the structure of Tic110, Tic110 from Cyanidioschyzon merolae (CmTic110) was characterized. We constructed three fragments, CmTic110_A, CmTic110_B and CmTic110_C, with increasing N‐terminal truncations, to perform small‐angle X‐ray scattering (SAXS) and X‐ray crystallography analyses and Dali structural comparison. Here we report the molecular envelope of CmTic110_B and CmTic110_C determined by SAXS, and the crystal structure of CmTic110_C at 4.2 Å. Our data indicate that the C‐terminal half of CmTic110 possesses a rod‐shaped helix‐repeat structure that is too flattened and elongated to be a channel. The structure is most similar to the HEAT‐repeat motif that functions as scaffolds for protein–protein interactions.     

Temperature effects on the hydrodynamic radius of the intrinsically disordered N‐terminal region of the p53 protein

Intrinsically disordered proteins (IDPs) are often characterized in terms of the hydrodynamic radius, R_h. The R_h of IDPs are known to depend on fractional proline content and net charge, where increased numbers of proline residues and increased net charge cause larger R_h. Though sequence and charge effects on the R_h of IDPs have been studied, the temperature sensitivity has been noted only briefly. Reported here are R_h measurements in the temperature range of 5–75°C for the intrinsically disordered N‐terminal region of the p53 protein, p53(1–93). Of note, the R_h of this protein fragment was highly sensitive to temperature, decreasing from 35 Å at 5°C to 26 Å at 75°C. Computer generated simulations of conformationally dynamic and disordered polypeptide chains were performed to provide a hypothesis for the heat‐induced compaction of p53(1–93) structure, which was opposite to the heat‐induced increase in R_h observed for a model folded protein. The simulations demonstrated that heat caused R_h to trend toward statistical coil values for both proteins, indicating that the effects of heat on p53(1–93) structure could be interpreted as thermal denaturation. The simulation data also predicted that proline content contributed minimally to the native R_h of p53(1–93), which was confirmed by measuring R_h for a substitution variant that had all 22 proline residues changed for glycine. Proteins 2014; 82:668–678. © 2013 Wiley Periodicals, Inc.