Dynamin and FtsZ. Missing links in mitochondrial and bacterial division.
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Animals↗
Biomedical subjects
H P Erickson
Publications and source records attributed to H P Erickson.
Ultrastructure and function of the fractalkine mucin domain in CX(3)C chemokine domain presentation.
Fractalkine (FKN), a CX(3)C chemokine/mucin hybrid molecule on endothelium, functions as an adhesion molecule to capture and induce firm adhesion of a subset of leukocytes in a selectin- and integrin-independent manner. We hypothesized that the FKN mucin domain may be important for its function in adhesion, and tested the ability of secreted alkaline phosphatase (SEAP) fusion proteins containing the entire extracellular region (FKN-SEAP), the chemokine domain (CX3C-SEAP), or the mucin domain (mucin-SEAP) to support firm adhesion under flow. CX3C-SEAP induced suboptimal firm adhesion of resting peripheral blood mononuclear cells, compared with FKN-SEAP, and mucin-SEAP induced no firm adhesion. CX3C-SEAP and FKN-SEAP bound to CX(3)CR1 with similar affinities. By electron microscopy, fractalkine was 29 nm in length with a long stalk (mucin domain), and a globular head (CX(3)C). To test the function of the mucin domain, a chimeric protein replacing the mucin domain with a rod-like segment of E-selectin was constructed. This chimeric protein gave the same adhesion of peripheral blood mononuclear cells as intact FKN, both when immobilized on glass and when expressed on the cell surface. This implies that the function of the mucin domain is to provide a stalk, extending the chemokine domain away from the endothelial cell surface to present it to flowing leukocytes.
Alkaline Phosphatase↗
Straight and curved conformations of FtsZ are regulated by GTP hydrolysis.
FtsZ assembles in vitro into protofilaments that can adopt two conformations-the straight conformation, which can assemble further into two-dimensional protofilament sheets, and the curved conformation, which forms minirings about 23 nm in diameter. Here, we describe the structure of FtsZ tubes, which are a variation of the curved conformation. In the tube the curved protofilament forms a shallow helix with a diameter of 23 nm and a pitch of 18 or 24 degrees. We suggest that this shallow helix is the relaxed structure of the curved protofilament in solution. We provide evidence that GTP favors the straight conformation while GDP favors the curved conformation. In particular, exclusively straight protofilaments and protofilament sheets are assembled in GMPCPP, a nonhydrolyzable GTP analog, or in GTP following chelation of Mg, which blocks GTP hydrolysis. Assembly in GDP produces exclusively tubes. The transition from straight protofilaments to the curved conformation may provide a mechanism whereby the energy of GTP hydrolysis is used to generate force for the constriction of the FtsZ ring in cell division.
Actin Cytoskeleton↗
The compact conformation of fibronectin is determined by intramolecular ionic interactions.
Fibronectin exists in a compact or extended conformation, depending upon environmental pH and salt concentration. Using recombinant fragments expressed in bacteria and baculovirus, we determined the domains responsible for producing fibronectin's compact conformation. Our velocity and equilibrium sedimentation data show that FN2-14 (a protein containing FN-III domains 2 through 14) forms dimers in low salt. Experiments with smaller fragments indicates that the compact conformation is produced by binding of FN12-14 of one subunit to FN2-3 of the other subunit in the dimer. The binding is weakened at higher salt concentrations, implying an electrostatic interaction. Furthermore, segment FN7-14+A, which contains the alternatively spliced A domain between FN11 and 12, forms dimers, whereas FN7-14 without A does not. Segment FN12-14+A also forms dimers, but the isolated A domain does not. These data imply an association of domain A with FN12-14, and the presence of A may favor an open conformation by competing with FN2-3 for binding to FN12-14.
Animals↗
Dynamics and elasticity of the fibronectin matrix in living cell culture visualized by fibronectin-green fluorescent protein.
Fibronectin (FN) forms the primitive fibrillar matrix in both embryos and healing wounds. To study the matrix in living cell cultures, we have constructed a cell line that secretes FN molecules chimeric with green fluorescent protein. These FN-green fluorescent protein molecules were assembled into a typical matrix that was easily visualized by fluorescence over periods of several hours. FN fibrils remained mostly straight, and they were seen to extend and contract to accommodate movements of the cells, indicating that they are elastic. When fibrils were broken or detached from cells, they contracted to less than one-fourth of their extended length, demonstrating that they are highly stretched in the living culture. Previous work from other laboratories has suggested that cryptic sites for FN assembly may be exposed by tension on FN. Our results show directly that FN matrix fibrils are not only under tension but are also highly stretched. This stretched state of FN is an obvious candidate for exposing the cryptic assembly sites.
Amino Acid Sequence↗
Oligomeric structure and tissue distribution of ficolins from mouse, pig and human.
Mouse plasma ficolin was purified by GlcNAc affinity and anion-exchange chromatography. Gel-filtration chromatography and gradient sedimentation indicated that mouse plasma ficolin is a 12-mer of approximately 35 kDa subunits, and electron microscopy showed the same parachute-like structure previously characterized for the pig ficolin 12-mer. Whereas the predominant form in pig plasma is a 24-mer, mouse and human plasma ficolin showed only the 12-mer form. We conclude that mouse plasma ficolin corresponds to the recently described ficolin A. We have identified a second mouse ficolin gene, ficolin B, which means that pig and mouse each have two ficolin genes, and human has three. One ficolin gene in all species is expressed in liver and is the primary source of plasma ficolin. Expression of this gene in other tissues, and expression of the second ficolin gene, appears to vary in different species.
Amidohydrolases↗
Ultrastructural and biochemical properties of the 120-kDa form of chick kinectin.
Kinectin, an integral membrane protein (160 kDa), was identified as a kinesin-binding protein. Analysis of the predicted amino acid sequence of kinectin cDNA indicated an alpha-helical coiled-coil structure from amino acid 320 to 1310. A 120-kDa kinectin has been observed consistently, and N-terminal sequencing showed that 232 amino acids were missing from the N terminus of full-length kinectin. 120-kDa kinectin was distributed in the supernatant and a low density fraction of vesicles, whereas both forms were in the high density fraction of vesicles. In the electron microscope, the 120-kDa form appeared as a linear molecule of 133 nm in length. In hydrodynamic studies, the cytosolic 120-kDa kinectin was a dimer. Monoclonal antibody molecules (anti-kinectin KR160.9) bound asymmetrically to kinectin often with two antibodies/kinectin, indicative of a parallel coiled-coil. Metabolic labeling with [3H]myristic acid showed that both the 120- and 160-kDa kinectin are myristoylated in chick embryo fibroblasts. The myristoylation of 120-kDa kinectin may provide a mechanism for linking it to a low density fraction of vesicles. Immunoprecipitation with a 160-kDa kinectin-specific antibody brought down the 120-kDa kinectin. Thus, we suggest that kinectin is an extended parallel coiled-coil dimer, often a heterodimer.
Amino Acid Sequence↗
The symmetrical structure of structural maintenance of chromosomes (SMC) and MukB proteins: long, antiparallel coiled coils, folded at a flexible hinge.
Structural maintenance of chromosomes (SMC) proteins function in chromosome condensation and several other aspects of DNA processing. They are large proteins characterized by an NH2-terminal nucleotide triphosphate (NTP)-binding domain, two long segments of coiled coil separated by a hinge, and a COOH-terminal domain. Here, we have visualized by EM the SMC protein from Bacillus subtilis (BsSMC) and MukB from Escherichia coli, which we argue is a divergent SMC protein. Both BsSMC and MukB show two thin rods with globular domains at the ends emerging from the hinge. The hinge appears to be quite flexible: the arms can open up to 180 degrees, separating the terminal domains by 100 nm, or close to near 0 degrees, bringing the terminal globular domains together. A surprising observation is that the approximately 300-amino acid-long coiled coils are in an antiparallel arrangement. Known coiled coils are almost all parallel, and the longest antiparallel coiled coils known previously are 35-45 amino acids long. This antiparallel arrangement produces a symmetrical molecule with both an NH2- and a COOH-terminal domain at each end. The SMC molecule therefore has two complete and identical functional domains at the ends of the long arms. The bifunctional symmetry and a possible scissoring action at the hinge should provide unique biomechanical properties to the SMC proteins.
Bacterial Proteins↗
The molecular elasticity of the extracellular matrix protein tenascin.
Extracellular matrix proteins are thought to provide a rigid mechanical anchor that supports and guides migrating and rolling cells. Here we examine the mechanical properties of the extracellular matrix protein tenascin by using atomic-force-microscopy techniques. Our results indicate that tenascin is an elastic protein. Single molecules of tenascin could be stretched to several times their resting length. Force-extension curves showed a saw-tooth pattern, with peaks of force at 137pN. These peaks were approximately 25 nm apart. Similar results have been obtained by study of titin. We also found similar results by studying recombinant tenascin fragments encompassing the 15 fibronectin type III domains of tenascin. This indicates that the extensibility of tenascin may be due to the stretch-induced unfolding of its fibronectin type III domains. Refolding of tenascin after stretching, observed when the force was reduced to near zero, showed a double-exponential recovery with time constants of 42 domains refolded per second and 0.5 domains per second. The former speed of refolding is more than twice as fast as any previously reported speed of refolding of a fibronectin type III domain. We suggest that the extensibility of the modular fibronectin type III region may be important in allowing tenascin-ligand bonds to persist over long extensions. These properties of fibronectin type III modules may be of widespread use in extracellular proteins containing such domain.
Alternative Splicing↗
Utilization of a soluble integrin-alkaline phosphatase chimera to characterize integrin alpha 8 beta 1 receptor interactions with tenascin: murine alpha 8 beta 1 binds to the RGD site in tenascin-C fragments, but not to native tenascin-C.
The integrin alpha 8 beta 1 has been reported to bind to fibronectin, vitronectin, and tenascin-C in cell adhesion or neurite outgrowth assays. Here, we describe cDNA cloning of the murine alpha 8 subunit, purification of a recombinant soluble heterodimer consisting of the extracellular domains of the murine alpha 8 and beta1 subunits, and development of a sensitive binding assay using a modified form of this heterodimer fused to alkaline phosphatase (AP). In binding assays, the purified alpha 8 beta 1-AP chimera exhibited the same divalent ion requirements for activation and binding specificity as cell surface alpha 8 beta 1: in the presence of Mn2+ it bound to fibronectin and vitronectin in an RGDS-peptide inhibitable manner. Contrary to previous reports, we found no evidence that alpha 8 beta 1, expressed on K562 cells or as an AP chimera, interacts strongly with native tenascin-C. In binding, adhesion, and spreading assays, significant interactions were observed only to short fragments of tenascin-C containing the third fibronectin type III repeat which contains an RGD sequence. Full length tenascin-C and longer fragments containing this repeat did not appear to serve as ligands, implying that the RGD site in native tenascin-C is a cryptic binding site for this integrin, exposed by removal of adjacent domains. Soluble integrin-AP chimeras should be generally useful for identifying and characterizing integrin interactions with ligands.
Alkaline Phosphatase↗
FtsZ from Escherichia coli, Azotobacter vinelandii, and Thermotoga maritima--quantitation, GTP hydrolysis, and assembly.
We have cloned the ftsZ genes from Thermotoga maritima and Azotobacter vinelandii and expressed the proteins (TmFtsZ and AzFtsZ) in Escherichia coli. We compared these proteins to E. coli FtsZ (EcFtsZ), and found that several remarkable features of their GTPase activities were similar for all three species, implying that these characteristics may be universal among FtsZs. Using a calibrated protein assay, we found that all three FtsZs bound 1 mole guanine nucleotide per mole FtsZ and hydrolyzed GTP at high rates (> 2 GTP per FtsZ per min). All three required magnesium and a monovalent cation for GTP hydrolysis. Previous reports showed that EcFtsZ (and some other species) required potassium. We confirmed this specificity for EcFtsZ but found that potassium and sodium both worked for Az- and TmFtsZ. Specific GTPase activity had a striking dependence on FtsZ concentration: activity (per FtsZ molecule) was absent or low below 50 microg/ml, rose steeply from 50 to 300 microg/ml and plateaued at a constant high value above 300 microg/ml. This finding suggests that the active state requires a polymer that is assembled cooperatively at 50-300 microg/ml. A good candidate for the active polymer was visualized by negative stain electron microscopy--straight protofilaments and protofilament pairs were seen under all conditions with active GTPase. We suggest that the GTP hydrolysis of FtsZ may be coupled to assembly, as it is for tubulin, with hydrolysis occurring shortly after an FtsZ monomer associates onto a protofilament end. As a part of this study, we determined the concentration of EcFtsZ and TmFtsZ by quantitative amino acid analysis and used this to standardize the bicinchonic acid colorimetric assay. This is the first accurate determination of FtsZ concentration. Using this standard and quantitative Western blotting, we determined that the average E. coli cell has 15,000 molecules of FtsZ, at a concentration of 400 microg/ml. This is just above the plateau for full GTPase activity in vitro.
Actin Cytoskeleton↗
Purification and assembly of FtsZ.
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Ammonium Sulfate↗
Mitogenic and adhesive effects of tenascin-C on human hematopoietic cells are mediated by various functional domains.
In the adult organism, the extracellular matrix molecule tenascin-C is prominently expressed in the bone marrow. Bone marrow mononuclear cells can adhere to plastic-immobilized tenascin-C, and in the present study we have used bacterial expression proteins to map the domains of tenascin-C responsible for binding of hematopoietic cells. A strong binding site was found to be located within the fibrinogen-like domain, and this binding could be inhibited by heparin, suggesting interactions with membrane-bound heparan sulfate proteoglycans. A second strong binding site was identified within the fibronectin type III-like repeats 6-8, and was also inhibitable by heparin. Adhesion to both attachment sites could not be blocked by various anti-integrin antibodies. A third hematopoietic cell binding site is located in the fibronectin type III-like repeats 1-5, which harbor an RGD sequence in the third fibronectin type III-like repeat. Binding to this domain, however, seems to be RGD-independent, since RGD-containing peptides could not inhibit cell binding; the addition of heparin also did not block adhesion to this domain. Since contradictory results had been reported on a proliferative effect of soluble tenascin-C, we also analyzed its activity on hematopoietic cells. The heterogeneous bone marrow mononuclear cells show a striking proliferative response in the presence of tenascin-C which is concentration-dependent. This result indicates a strong mitogenic activity of tenascin-C on primary hematopoietic cells. Using recombinant fragments of human tenascin-C, we identified several mitogenic domains within the tenascin-C molecule. These adhesive and mitogenic effects of tenascin-C suggest a direct functional association with proliferation and differentiation of hematopoietic cells within the bone marrow microenvironment.
Binding Sites↗
Atomic structures of tubulin and FtsZ.
The recently published atomic structures of tubulin and FtsZ are a research milestone. The N-terminal GTP-binding domains of tubulin and FtsZ are virtually identical in structure, as expected from the substantial sequence identity. Sequence identity is absent from the C-terminal domains, but they also have virtually identical structures. A surprising finding is that the N-terminal GTP-binding domain is structurally homologous to that of Ras and other G proteins, despite the completely different GTP-binding sequence motifs. This article discusses these findings and the molecular mechanisms that can now be addressed with the atomic structures.
Amino Acid Sequence↗
Pervasive conformational fluctuations on microsecond time scales in a fibronectin type III domain.
A novel off-resonance rotating-frame 15N NMR spin relaxation experiment is used to characterize conformational fluctuations with correlation times between 32 and 175 microseconds in the third fibronectin type III domain of human tenascin-C. Conformational fluctuations of contiguous regions of the beta-sandwich structure of the type III domain may represent collective motions, such as transient twisting or breathing of the beta-sheets. Flexibility of the loop containing the Arg-Gly-Asp (RGD) tripeptide may affect the accessibility of this motif in protein-protein interactions.
Diffusion↗
Soluble monomeric P-selectin containing only the lectin and epidermal growth factor domains binds to P-selectin glycoprotein ligand-1 on leukocytes.
Under shear stress, leukocytes use P-selectin glycoprotein ligand-1 (PSGL-1) to tether to and roll on P-selectin expressed on activated platelets or endothelial cells. P-selectin has an NH2-terminal lectin domain, an epidermal growth factor (EGF)-like motif, nine consensus repeats (CRs), a transmembrane domain, and a cytoplasmic tail. To determine whether the CRs are required for P-selectin to bind PSGL-1, we expressed a soluble protein (Lec-EGF) that contained only the lectin and EGF domains, plus a short C-terminal epitope tag. Electron microscopy and hydrodynamic analysis confirmed that Lec-EGF was monomeric, as previously shown for soluble P-selectin (sPS) that contained the lectin and EGF domains plus all nine CRs. Fluid-phase Lec-EGF or sPS inhibited binding of oligomeric125I-labeled membrane-derived P-selectin (mPS) to PSGL-1 on neutrophils and binding of 125I-PSGL-1 to immobilized mPS. The IC50 for inhibiting binding of mPS to neutrophils was fivefold greater for Lec-EGF than for sPS, whereas the IC50 for inhibiting binding of mPS to purified PSGL-1 was indistinguishable for Lec-EGF and sPS. Under static or shear conditions, neutrophils used PSGL-1 to tether to or roll on Lec-EGF that was captured by an immobilized monoclonal antibody to the C-terminal epitope. These data show that P-selectin requires only the lectin and EGF domains to bind to PSGL-1.
Cell Adhesion↗
Backbone dynamics of homologous fibronectin type III cell adhesion domains from fibronectin and tenascin.
BACKGROUND: Fibronectin type III domains are found as autonomously-folded domains in a large variety of multidomain proteins, including extracellular matrix proteins. A subset of these domains employ an Arg-Gly-Asp (RGD) tripeptide motif to mediate contact with cell-surface receptors (integrins). This motif mediates protein-protein interactions in a diverse range of biological processes, such as in tissue development, would healing and metastasis. The molecular basis for affinity and specificity of cell adhesion via type III domains has not been clearly established. The tenth type III domain from fibronectin (FNfn10) and the third type III domain from tenascin-C (TNfn3) have 27% sequence identity and share the same overall protein fold, but present the RGD motifs in different structural contexts. The dynamical properties of the RGD motifs may affect the specificity and affinity of the FNfn10 and TNfn3 domains. Structure-dynamics correlations for these structurally homologous proteins may reveal common molecular features which are important to the dynamical properties of proteins. RESULTS: The intramolecular dynamics of the protein backbones of FNfn10 and TNfn3 have been studied by 15N nuclear spin relaxation. The FG loop in FNfn10, which contains the RGD motif, exhibits extensive flexibility on picosecond to nanosecond timescales, but motions on microsecond to millisecond timescales are not observed. The equivalent region in TNfn3 is as rigid as regular elements of secondary structure. The CC' loop also is more flexible on picosecond-nanosecond timescales in FNfn10 than in TNfn3. Conformational exchange, reflecting flexibility on microsecond-millisecond timescales, is observed in beta strands A and B of both FNfn10 and TNfn3. CONCLUSIONS: Comparison of the structures of the FNfn10 and TNfn3 reveals several features related to their different dynamical properties. The larger amplitude motions of loops in FNfn10 are consistent with the hypothesis that flexibility of these regions facilitates induced-fit recognition of fibronectin by multiple receptors. Similarly, the more rigid loops of TNfn3 may reflect greater specificity for particular integrins. The correlations observed between structural features and dynamical properties of the homologous type III domains indicate the influence of hydrogen bonding and hydrophobic packing on dynamical fluctuations in proteins.
Amino Acid Sequence↗
Two oligomeric forms of plasma ficolin have differential lectin activity.
Ficolins are plasma proteins with binding activity for carbohydrates, elastin, and corticosteroids. The ficolin polypeptide has a collagen-like domain that presumably brings three subunits together in a triple helical rod, a C-terminal fibrinogen-like domain (fbg) similar to that of tenascin, which presumably has the binding activities, and a small N-terminal domain that we find to be the primary site for forming the ficolin oligomer. By sedimentation equilibrium we determined that the main plasma form, which we call big ficolin, had mass of 827,000 Da, consistent with 24 subunits. Little ficolin, about half this size, was obtained after binding to a GlcNAc affinity column. Electron microscopy of little ficolin showed a parachute-like structure, with a small globe at one end, corresponding to the 12 N-terminal domains, and the fbg domains clustered together at the ends of the collagen rods. Big ficolin was formed by the face to face fusion of the fbg domains of two little ficolins, leaving the rods and N-terminal domains projecting at opposite ends. Little ficolin maintained a high affinity for the GlcNAc column, and big ficolin had a low affinity or none. The binding sites for ligands may be obscured in this big ficolin oligomer, providing a regulation of their activity.
Animals↗