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Richard F Collins

Publications and source records attributed to Richard F Collins.

17 recordsLinked to original sources
PubMed2006

Three-dimensional structure determination of a protein supercomplex that oxidizes methane to formaldehyde in Methylococcus capsulatus (Bath).

The oxidation of methane to methanol in methanotrophs is catalyzed by the enzyme methane monooxygenase (MMO). Two distinct forms of this enzyme exist, a soluble cytoplasmic MMO (sMMO) and a membrane-bound particulate form (pMMO). The active protein complex termed pMMO-C was purified recently from Methylococcus capsulatus (Bath). The complex consists of pMMO hydroxylase and an additional component pMMO-R, which was proposed to be the reductase for the pMMO complex. Further study of this complex has led here to the proposal that the pMMO-R is in fact methanol dehydrogenase, the subsequent enzyme in the methane oxidation pathway by methanotrophs. We describe here the biochemical and biophysical characterization of a stable purified complex of pMMO hydroxylase (pMMO-H) with methanol dehydrogenase (MDH) and report the first three-dimensional (3D) structure, determined by cryoelectron microscopy and single particle analysis to approximately 16 A resolution. The 3D structure reported here provides the first insights into the supramolecular organization of pMMO with MDH. These studies of pMMO-MDH complexes have provided further understanding of the structural basis for the particular functions of the enzymes in this system which might also be of relevance to the complete process of methane oxidation by methanotrophs under high copper concentration in the environment.

Bacterial Proteins↗
PubMed2006

Structural variation and immune recognition of the P1.2 subtype meningococcal antigen.

Neisseria meningitidis is a globally important cause of bacterial meningitis and septicemia. No comprehensive antimeningococcal vaccine is available, largely as a consequence of the high sequence diversity of those surface proteins that could function as components of a vaccine. One such component is the protein PorA, a major surface porin of this Gram-negative organism that has been used in a number of experimental and licensed vaccines. Here we describe a series of experiments designed to investigate the consequences for antibody recognition of sequence diversity within a PorA antigen. The binding of a 14-residue peptide, corresponding to the P1.2 subtype antigen, to the MN16C13F4 monoclonal antibody was sensitive to mutation of five out of the six residues within the epitope sequence. The crystal structure of the antibody Fab fragment, determined in complex with the peptide antigen, shows a remarkably hydrophobic binding site and interactions between the antigen and antibody are dominated by apolar residues. Nine intrachain hydrogen bonds are formed within the antigen which maintain the beta-hairpin conformation of the peptide. These hydrogen bonds involve residues that are highly conserved amongst different P1.2 sequence variants, suggesting that some positions may be conserved for structural reasons in these highly polymorphic regions. The sensitivity of antibody recognition of the antigen towards mutation provides a structural explanation for the widespread sequence variation seen in different PorA sequences in this region. Single point mutations are sufficient to remove binding capability, providing a rationale for the manner in which different meningococcal PorA escape variants arise.

Amino Acid Sequence↗
PubMed2006

Purification and 3D structural analysis of oligomeric human multidrug transporter ABCG2.

ABCG2 is a multidrug efflux pump associated with resistance of cancer cells to a plethora of unrelated drugs. ABCG2 is a "half-transporter," and previous studies have indicated that it forms homodimers and higher oligomeric species. In this manuscript, electron microscopic structural analysis directly addressed this issue. An N-terminal hexahistidine-tagged ABCG2(R482G) isoform was expressed to high levels in insect cells. An extensive detergent screen was employed to effect extraction of ABCG2(R482G) from membranes and identified only the fos-choline detergents as efficient. Soluble protein was purified to >95% homogeneity by a three-step procedure while retaining the ability to bind substrates. Cryonegative stain electron microscopy of purified ABCG2(R482G) provided 3D structural data at a resolution of approximately 18 A. Single-particle analysis revealed that the complex forms a tetrameric complex ( approximately 180 A in diameter x approximately 140 A high) with an aqueous central region. We interpret the tetrameric structure as comprising four homodimeric ABCG2(R482G) complexes.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2006

Topology of the outer-membrane secretin PilQ from Neisseria meningitidis.

Neisseria meningitidis is the causative agent of epidemic meningococcal meningitis and septicaemia. Type IV pili are surface organelles that mediate a variety of functions, including adhesion, twitching motility, and competence for DNA binding and uptake in transformation. The secretin PilQ is required for type IV pilus expression at the cell surface, and forms a dodecameric cage-like macromolecular complex in the meningococcal outer membrane. PilQ-null mutants are devoid of surface pili, and prevailing evidence suggests that the PilQ complex facilitates extrusion and retraction of type IV pili across the outer membrane. Defining the orientation of the meningococcal PilQ complex in the membrane is a prerequisite for understanding the structure-function relationships of this important protein in pilus biology. In order to begin to define the topology of the PilQ complex in the outer membrane, polyhistidine insertions in N- and C-terminal regions of PilQ were constructed, and their subcellular locations examined. Notably, the insertion epitopes at residues 205 and 678 were located within the periplasm, whereas residue 656 was exposed at the outer surface of the outer membrane. Using electron microscopy with Ni-NTA gold labelling, it was demonstrated that the insertion at residue 205 within the N-terminus mapped to a site on the arm-like features of the 3D structure of the PilQ multimer. Interestingly, mutation of the same region gave rise to an increase in vancomycin permeability through the PilQ complex. The results yield novel information on the PilQ N-terminal location and function in the periplasm, and reveal a complex organization of the membrane-spanning secretin in vivo.

Bacterial Outer Membrane Proteins↗
PubMed2005

3-D structural and functional characterization of the purified KATP channel complex Kir6.2-SUR1.

ATP-sensitive potassium (K(ATP)) channels conduct potassium ions across cell membranes and thereby couple cellular energy metabolism to membrane electrical activity. Here, we report the heterologous expression and purification of a functionally active K(ATP) channel complex composed of pore-forming Kir6.2 and regulatory SUR1 subunits, and determination of its structure at 18 A resolution by single-particle electron microscopy. The purified channel shows ATP-ase activity similar to that of ATP-binding cassette proteins related to SUR1, and supports Rb(+) fluxes when reconstituted into liposomes. It has a compact structure, with four SUR1 subunits embracing a central Kir6.2 tetramer in both transmembrane and cytosolic domains. A cleft between adjacent SUR1s provides a route by which ATP may access its binding site on Kir6.2. The nucleotide-binding domains of adjacent SUR1 appear to interact, and form a large docking platform for cytosolic proteins. The structure, in combination with molecular modelling, suggests how SUR1 interacts with Kir6.2.

ATP-Binding Cassette Transporters↗
PubMed2005

Periplasmic protein-protein contacts in the inner membrane protein Wzc form a tetrameric complex required for the assembly of Escherichia coli group 1 capsules.

The K antigenic capsular polysaccharide forms a structural layer, the capsule, on the surfaces of Escherichia coli cells. The capsule provides an important protective covering that helps protect encapsulated bacteria from host immune defenses. The assembly and translocation of the capsule requires proteins in the inner and outer membranes. The inner membrane protein Wzc is a tyrosine autokinase that plays an essential role in what is believed to be a coordinated biosynthesis and secretion process. Mutants lacking Wzc can form K antigen oligosaccharides but are unable to polymerize high molecular weight capsular polymers. Wzc homologs have been identified in exopolymer biosynthesis systems in many different Gram-negative and -positive bacteria. Using single particle averaging on cryo-negatively stained samples, we have produced the first three-dimensional structure of this type of membrane protein in its phosphorylated state at approximately 14 A resolution. Perfluoro-octanoate-PAGE analysis of detergent-solubilized oligomeric Wzc and symmetry analysis of the transmission electron microscopy data clearly demonstrated that Wzc forms a tetrameric complex with C4 rotational symmetry. Viewed from the top of the complex, the oligomer is square with a diameter of approximately 100 A and can be divided into four separate densities. From the side, Wzc is approximately 110 A high and has a distinctive appearance similar to an extracted molar tooth. The upper "crown" region is approximately 55 A high and forms a continuous ring of density. Four unconnected "roots" ( approximately 65 A high) emerge from the underside of the crown. We propose that the crown is formed by protein-protein contacts from the four Wzc periplasmic domains, while each root represents an individual cytoplasmic tyrosine autokinase domain.

Bacterial Proteins↗
PubMed2005

Coronin-1 function is required for phagosome formation.

Coronin-1 is an actin-associated protein whose function in actin dynamics has remained obscure. All coronin proteins have a variable N-terminal domain, followed by WD repeats and a C-terminal coiled-coil dimerization domain. Transfection of coronin-1-GFP into RAW 264.7 cells revealed that coronin rapidly and transiently associates with the phagosome. To determine if coronin is involved in mammalian phagocytosis we used a dominant-negative approach by expressing only the central WD domains. However, this caused cell rounding and dissociation from the substratum, hampering analysis of their phenotype. We therefore developed TAT-fusion constructs of coronin-1 WD domains to acutely introduce the recombinant protein fragment into live cells. We show that although TAT-WD has no effect on binding of opsonized RBCs to RAW 264.7 cells, receptor clustering or several downstream signaling events, lamellipodial extensions, and actin accumulation at the base of the bound particle were diminished. Furthermore, Arp3 accumulation at the phagosome was impaired after TAT-WD treatment. Interestingly, whereas coronin-1 also accumulates at the sites of actin remodeling associated with Salmonella invasion, TAT-WD had no effect on this process. Together, our data demonstrates that coronin-1 is required for an early step in phagosome formation, consistent with a role in actin polymerization.

Actins↗
PubMed2005

Interaction with type IV pili induces structural changes in the bacterial outer membrane secretin PilQ.

Type IV pili are cell surface organelles found on many Gram-negative bacteria. They mediate a variety of functions, including adhesion, twitching motility, and competence for DNA uptake. The type IV pilus is a helical polymer of pilin protein subunits and is capable of rapid polymerization or depolymerization, generating large motor forces in the process. Here we show that a specific interaction between the outer membrane secretin PilQ and the type IV pilus fiber can be detected by far-Western analysis and sucrose density gradient centrifugation. Transmission electron microscopy of preparations of purified pili, to which the purified PilQ oligomer had been added, showed that PilQ was uniquely located at one end of the pilus fiber, effectively forming a "mallet-type" structure. Determination of the three-dimensional structure of the PilQ-type IV pilus complex at 26-angstroms resolution showed that the cavity within the protein complex was filled. Comparison with a previously determined structure of PilQ at 12-angstroms resolution indicated that binding of the pilus fiber induced a dissociation of the "cap" feature and lateral movement of the "arms" of the PilQ oligomer. The results demonstrate that the PilQ structure exhibits a dynamic response to the binding of its transported substrate and suggest that the secretin could play an active role in type IV pilus assembly as well as secretion.

Blotting, Western↗
PubMed2004

Structure of the Neisseria meningitidis outer membrane PilQ secretin complex at 12 A resolution.

The bacterial pathogen Neisseria meningitidis expresses long, thin, retractile fibers (called type IV pili) from its cell surface and uses these adhesive structures to mediate primary attachment to epithelial cells during host colonization and invasion. PilQ is an outer membrane protein complex that is essential for the translocation of these pili across the outer membrane. Here, we present the structure of the PilQ complex determined by cryoelectron microscopy to 12 A resolution. The dominant feature of the structure is a large central cavity, formed by four arm features that spiral upwards from a squared ring base and meet to form a prominent cap region. The cavity, running through the center of the complex, is continuous and is effectively sealed at both the top and bottom. Analysis of the complex using self-orientation and by examination of two-dimensional crystals indicates a strong C4 rotational symmetry, with a much weaker C12 rotational symmetry, consistent with PilQ possessing true C4 symmetry with C12 quasi-symmetry. We therefore suggest that the complex is a homododecamer, formed by association of 12 PilQ polypeptide chains into a tetramer of trimers. The structure of the PilQ complex, with its large and well defined central chamber, suggests that it may not function solely as a passive portal in the outer membrane, but could be actively involved in mediating pilus assembly or modification.

Cryoelectron Microscopy↗
PubMed2004

Three-dimensional structure of Wza, the protein required for translocation of group 1 capsular polysaccharide across the outer membrane of Escherichia coli.

Wza is a highly conserved multimeric outer membrane protein complex required for the surface expression of the serotype K30 group 1 capsular polysaccharide in Escherichia coli. Here we present the first three-dimensional structure of this type of polysaccharide exporter at a 15.5-A resolution obtained using single particle averaging on a dataset of cryo-negatively stained protein. Previous structural studies on purified Wza have revealed a homo-oligomeric ring structure that is most probably composed of eight subunits. Symmetry analysis of the three-dimensional structure combined with biochemical two- and three-dimensional crystallographic data strongly suggest that Wza is an octameric complex with a C4 quasi-rotational symmetry and is organized as a tetramer of dimeric subunits. Wza is best described as a stack of two 4-A high rings with differing diameters providing a mushroom-like aspect from the side. The larger ring has a distinctive square shape with a diameter of 115 A, whereas the smaller is almost circular with a diameter of 90 A. In the center of the complex and enclosed by the four symmetrical arms is a small elliptical cagelike cavity of approximately 40 A in diameter. The central cavity is effectively sealed at the top and bottom of the complex but has small inter-arm holes when viewed from the side. We discuss the structure of this complex and implications in the surface translocation of cell-surface polysaccharide.

Bacterial Outer Membrane Proteins↗
PubMed2003

The three-dimensional structure of the cardiac L-type voltage-gated calcium channel: comparison with the skeletal muscle form reveals a common architectural motif.

We describe here the first three-dimensional structure of the cardiac L-type voltage-gated calcium channel (VGCC) purified from bovine heart. The structure was determined by electron microscopy and single particle analysis of negatively stained complexes, using the angular reconstitution method. The cardiac VGCC can be isolated as a stable dimer, as reported previously for the skeletal muscle VGCC, with a central aqueous chamber formed by the two halves of the complex. Moreover, we demonstrate that the dimeric cardiac VGCC binds the dihydropyridine [3H]azidopine with a Kd approximately 310 pM. We have compared the cardiac VGCC structure with the skeletal muscle form, determined using the same reconstructive methodology, allowing us to identify common and distinct features of the complexes. By using antibody and lectin-gold labeling, we have localized the intracellular beta polypeptides and the extracellular glycosylation sites of the skeletal muscle VGCC, which can be correlated to the cardiac three-dimensional structure. From the data presented here the assignment of the orientation of the VGCC complexes with respect to the lipid bilayer is now possible. A difference between the cardiac and skeletal muscle ion channels is apparent in the putative transmembrane region, which would be consistent with the absence of the gamma subunit in the cardiac VGCC assembly.

Amino Acid Motifs↗
PubMed2003

Three-dimensional structure of the Neisseria meningitidis secretin PilQ determined from negative-stain transmission electron microscopy.

The PilQ secretin from the pathogenic bacterium Neisseria meningitidis is an integral outer membrane protein complex which plays a crucial role in the biogenesis of type IV pili. We present here the first three-dimensional structure of this type of secretin at 2.5-nm resolution, obtained by single-particle averaging methods applied to the purified protein complex visualized in a negative stain. In projection, the PilQ complex is circular, with a donut-like appearance. When viewed from the side it has a rounded, conical profile. The complex was demonstrated to have 12-fold rotational symmetry, and this property was used to improve the quality of the density map by symmetry averaging. The dominant feature of the structure is a cavity, 10 nm deep, within the center of the molecule. The cavity is funnel-shaped in cross section, measures 6.5 nm in diameter at the top of the complex, and tapers to a closed point, effectively blocking formation of a continuous pore through the PilQ complex. These results suggest that the complex would have to undergo a conformational change in order to accommodate an assembled pilus fiber of diameter 6.5 nm running through the outer membrane.

Biological Transport↗
PubMed2002

Syntaxins 13 and 7 function at distinct steps during phagocytosis.

The phagosome is a dynamic organelle that undergoes progressive changes to acquire the machinery required to kill and degrade internalized foreign particles. This maturation process involves sequential interaction of newly formed phagosomes with several components of the endocytic pathway. The proteins that mediate the ordered fusion of endosomes and lysosomes with the phagosome are not known. In this study, we investigated the possible role of syntaxins present in the endo/lysosomal pathway in directing phagosomal maturation. We show that in phagocytic cells syntaxin 13 is localized to the recycling endosome compartment, while syntaxin 7 is found in late endosomes/lysosomes. Both proteins are recruited to the phagosome, but syntaxin 13 is acquired earlier and rapidly recycles off the phagosome, while syntaxin 7 is recruited later and continues to accumulate throughout the maturation process. Overexpression of truncated (cytosolic) forms of syntaxin 13 or 7 had no effect on phagocytosis, but exerted an inhibitory effect on phagosomal maturation. These results indicate that syntaxins 13 and 7 are both required for interaction of endosomes and/or lysosomes with the phagosome, but play distinct roles in the maturation process.

Animals↗
PubMed2002

Inhibition of phosphatidylinositol-4-phosphate 5-kinase Ialpha impairs localized actin remodeling and suppresses phagocytosis.

Actin polymerization drives the extension of pseudopods required for phagocytosis. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) is thought to play a central role in this process, because it interacts with several actin-regulatory proteins and undergoes acute and localized changes at sites of phagocytosis. We therefore studied whether phosphatidylinositol-4-phosphate 5-kinase (PIPK), the enzyme responsible for the generation of PIP(2) from phosphatidylinositol 4-phosphate, is involved in the control of phagocytosis. PIPKIalpha was found to accumulate transiently on forming phagosomes. To test the functional involvement of PIPKIalpha in particle engulfment, we generated a double mutant (D309N/R427Q) that lacks kinase activity. When ectopically expressed in cultured cells, this mutant is targeted to the plasma membrane and accumulates at the phagosomal cup during particle engulfment. Expression of PIP5KIalpha D309N/R427Q impaired phagocytosis in RAW264.7 macrophages and in engineered phagocytes generated by transfection of Fc receptors in Chinese hamster ovary cells. Inhibition of phagocytosis could not be attributed to defects in particle binding or receptor clustering, which was monitored using green fluorescent protein-tagged Fcgamma receptors. Instead, expression of the inactive kinase diminished the accumulation of PIP(2) and of F-actin in the phagosomal cup. These data suggest that PIPKIalpha activity is involved in the actin remodeling that is a prerequisite for efficient phagocytosis. PIPKIalpha appears to contribute to the transient changes in PIP(2) levels that are associated with, and likely required for, the recruitment and regulation of actin-modulating proteins.

Actins↗
PubMedDate not supplied

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Centers for Disease Control and Prevention, U.S.↗