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Gary S Shaw

Publications and source records attributed to Gary S Shaw.

At least 19 recordsLinked to original sources
PubMed2006

Insights into S100 target specificity examined by a new interaction between S100A11 and annexin A2.

S100 proteins are a group of EF-hand calcium-signaling proteins, many of which interact with members of the calcium- and phospholipid-binding annexin family of proteins. This calcium-sensitive interaction enables two neighboring membrane surfaces, complexed to different annexin proteins, to be brought into close proximity for membrane reorganization, using the S100 protein as a bridging molecule. S100A11 and S100A10 are two members of the S100 family found to interact with the N-termini of annexins A1 and A2, respectively. Despite the high degree of structural similarity between these two complexes and the sequences of the peptides, earlier studies have shown that there is little or no cross-reactivity between these two S100s and the annexin peptides. In the current work the specificity and the affinity of the interaction of the N-terminal sequences of annexins A1 and A2 with Ca2+-S100A11 were investigated. Through the use of alanine-scanning peptide array experiments and NMR spectroscopy, an approximate 5-fold tighter interaction was identified between Ca2+-S100A11 and annexin A2 (approximately 3 microM) compared to annexin A1 (approximately 15 microM). Chemical shift mapping revealed that the binding site for annexin A2 on S100A11 was similar to that observed for the annexin A1 but with distinct differences involving the C-terminus of the annexin A2 peptide. In addition, kinetic measurements based on NMR titration data showed that annexin A2 binding to Ca2+-S100A11 occurs at a comparable rate (approximately 120 s(-1)) to that observed for membrane fusion processes such as endo- and exocytosis.

Amino Acid Sequence↗
PubMed2006

Calcium-dependent and -independent interactions of the S100 protein family.

The S100 proteins comprise at least 25 members, forming the largest group of EF-hand signalling proteins in humans. Although the proteins are expressed in many tissues, each S100 protein has generally been shown to have a preference for expression in one particular tissue or cell type. Three-dimensional structures of several S100 family members have shown that the proteins assume a dimeric structure consisting of two EF-hand motifs per monomer. Calcium binding to these S100 proteins, with the exception of S100A10, results in an approx. 40 degrees alteration in the position of helix III, exposing a broad hydrophobic surface that enables the S100 proteins to interact with a variety of target proteins. More than 90 potential target proteins have been documented for the S100 proteins, including the cytoskeletal proteins tubulin, glial fibrillary acidic protein and F-actin, which have been identified mostly from in vitro experiments. In the last 5 years, efforts have concentrated on quantifying the protein interactions of the S100 proteins, identifying in vivo protein partners and understanding the molecular specificity for target protein interactions. Furthermore, the S100 proteins are the only EF-hand proteins that are known to form both homo- and hetero-dimers, and efforts are underway to determine the stabilities of these complexes and structural rationales for their formation and potential differences in their biological roles. This review highlights both the calcium-dependent and -independent interactions of the S100 proteins, with a focus on the structures of the complexes, differences and similarities in the strengths of the interactions, and preferences for homo- compared with hetero-dimeric S100 protein assembly.

Binding Sites↗
PubMed2006

Folding kinetics of the S100A11 protein dimer studied by time-resolved electrospray mass spectrometry and pulsed hydrogen-deuterium exchange.

This study reports the application of electrospray ionization (ESI) mass spectrometry (MS) with on-line rapid mixing for millisecond time-resolved studies of the refolding and assembly of a dimeric protein complex. Acid denaturation of S100A11 disrupts the native homodimeric protein structure. Circular dichroism and HSQC nuclear magnetic resonance measurements reveal that the monomeric subunits unfold to a moderate degree but retain a significant helicity and some tertiary structural elements. Following a rapid change in solution conditions to a slightly basic pH, the native protein reassembles with an effective rate constant of 6 s(-)(1). The ESI charge state distributions measured during the reaction suggest the presence of three kinetic species, namely, a relatively unfolded monomer (M(U)), a more tightly folded monomeric reaction intermediate (M(F)), and dimeric S100A11. These three forms exhibit distinct calcium binding properties, with very low metal loading levels for M(U), up to two calcium ions for M(F), and up to four for the dimer. Surprisingly, on-line pulsed hydrogen-deuterium exchange (HDX) reveals that each of the monomeric forms of the protein comprises two subspecies that can be distinguished on the basis of their isotope exchange levels. As the reaction proceeds, the more extensively labeled species are depleted. The exponential nature of the measured intensity-time profiles implies that the rate-determining step of the overall process is a unimolecular event. The kinetics are consistent with a sequential folding and assembly mechanism involving two increasingly nativelike monomeric intermediates en route to the native S100A11 dimer.

Animals↗
PubMed2005

Solution structure of the Escherichia coli protein ydhR: a putative mono-oxygenase.

YdhR is a 101-residue conserved protein from Escherichia coli. Sequence searches reveal that the protein has >50% identity to proteins found in a variety of other bacterial genomes. Using size exclusion chromatography and fluorescence spectroscopy, we determined that ydhR exists in a dimeric state with a dissociation constant of approximately 40 nM. The three-dimensional structure of dimeric ydhR was determined using NMR spectroscopy. A total of 3400 unambiguous NOEs, both manually and automatically assigned, were used for the structure calculation that was refined using an explicit hydration shell. A family of 20 structures was obtained with a backbone RMSD of 0.48 A for elements of secondary structure. The structure reveals a dimeric alpha,beta fold characteristic of the alpha+beta barrel superfamily of proteins. Bioinformatic approaches were used to show that ydhR likely belongs to a recently identified group of mono-oxygenase proteins that includes ActVA-Orf6 and YgiN and are involved in the oxygenation of polyaromatic ring compounds.

Amino Acid Sequence↗
PubMed2005

Ubiquitin manipulation by an E2 conjugating enzyme using a novel covalent intermediate.

Degradation of misfolded and damaged proteins by the 26 S proteasome requires the substrate to be tagged with a polyubiquitin chain. Assembly of polyubiquitin chains and subsequent substrate labeling potentially involves three enzymes, an E1, E2, and E3. E2 proteins are key enzymes and form a thioester intermediate through their catalytic cysteine with the C-terminal glycine (Gly76) of ubiquitin. This thioester intermediate is easily hydrolyzed in vitro and has eluded structural characterization. To overcome this, we have engineered a novel ubiquitin-E2 disulfide-linked complex by mutating Gly76 to Cys76 in ubiquitin. Reaction of Ubc1, an E2 from Saccharomyces cerevisiae, with this mutant ubiquitin resulted in an ubiquitin-E2 disulfide that could be purified and was stable for several weeks. Chemical shift perturbation analysis of the disulfide ubiquitin-Ubc1 complex by NMR spectroscopy reveals an ubiquitin-Ubc1 interface similar to that for the ubiquitin-E2 thioester. In addition to the typical E2 catalytic domain, Ubc1 contains an ubiquitin-associated (UBA) domain, and we have utilized NMR spectroscopy to demonstrate that in this disulfide complex the UBA domain is freely accessible to non-covalently bind a second molecule of ubiquitin. The ability of the Ubc1 to bind two ubiquitin molecules suggests that the UBA domain does not interact with the thioester-bound ubiquitin during polyubiquitin chain formation. Thus, construction of this novel ubiquitin-E2 disulfide provides a method to characterize structurally the first step in polyubiquitin chain assembly by Ubc1 and its related class II enzymes.

Catalytic Domain↗
PubMed2004

Solution structure of the flexible class II ubiquitin-conjugating enzyme Ubc1 provides insights for polyubiquitin chain assembly.

E2 conjugating enzymes form a thiol ester intermediate with ubiquitin, which is subsequently transferred to a substrate protein targeted for degradation. While all E2 proteins comprise a catalytic domain where the thiol ester is formed, several E2s (class II) have C-terminal extensions proposed to control substrate recognition, dimerization, or polyubiquitin chain formation. Here we present the novel solution structure of the class II E2 conjugating enzyme Ubc1 from Saccharomyces cerevisiae. The structure shows the N-terminal catalytic domain adopts an alpha/beta fold typical of other E2 proteins. This domain is physically separated from its C-terminal domain by a 22-residue flexible tether. The C-terminal domain adopts a three-helix bundle that we have identified as an ubiquitin-associated domain (UBA). NMR chemical shift perturbation experiments show this UBA domain interacts in a regioselective manner with ubiquitin. This two-domain structure of Ubc1 was used to identify other UBA-containing class II E2 proteins, including human E2-25K, that likely have a similar architecture and to determine the role of the UBA domain in facilitating polyubiquitin chain formation.

Amino Acid Sequence↗
PubMed2004

Solution NMR structure and X-ray absorption analysis of the C-terminal zinc-binding domain of the SecA ATPase.

The solution NMR structure of a 22-residue Zn(2+)-binding domain (ZBD) from Esherichia coli preprotein translocase subunit SecA is presented. In conjunction with X-ray absorption analysis, the NMR structure shows that three cysteines and a histidine in the sequence CXCXSGX(8)CH assume a tetrahedral arrangement around the Zn(2+) atom, with an average Zn(2+)-S bond distance of 2.30 A and a Zn(2+)-N bond distance of 2.03 A. The NMR structure shows that ND1 of His20 binds to the Zn(2+) atom. The ND1-Zn(2+) bond is somewhat strained: it makes an angle of approximately 17 degrees with the plane of the ring, and it also shows a significant "in-plane" distortion of 13 degrees. A comprehensive sequence alignment of the SecA-ZBD from many different organisms shows that, along with the four Zn(2+) ligands, there is a serine residue (Ser12) that is completely conserved. The NMR structure indicates that the side chain of this serine residue forms a strong hydrogen bond with the thiolate of the third cysteine residue (Cys19); therefore, the conserved serine appears to have a critical role in the structure. SecB, an export-specific chaperone, is the only known binding partner for the SecA-ZBD. A phylogenetic analysis using 86 microbial genomes shows that 59 of the organisms carry SecA with a ZBD, but only 31 of these organisms also possess a gene for SecB, indicating that there may be uncharacterized binding partners for the SecA-ZBD.

Adenosine Triphosphatases↗
PubMed2004

Human S100B protein interacts with the Escherichia coli division protein FtsZ in a calcium-sensitive manner.

S100B is a small, dimeric EF-hand calcium-binding protein abundant in vertebrates. Upon calcium binding, S100B undergoes a conformational change allowing it to interact with a variety of target proteins, including the cytoskeletal proteins tubulin and glial fibrillary acidic protein. In both cases, S100B promotes the in vitro disassembly of these proteins in a calcium-sensitive manner. Despite this, there is little in vivo evidence for the interaction of proteins such as tubulin with S100B. To probe these interactions, we studied the expression of human S100B in Escherichia coli and its interaction with the prokaryotic ancestor of tubulin, FtsZ, the major protein involved in bacterial division. Expression of S100B protein in E. coli results in little change in FtsZ protein levels, causes a filamenting bacterial phenotype characteristic of FtsZ inhibition, and leads to missed rounds of cell division. Further, S100B localizes to positions similar to those of FtsZ in bacterial filaments: the small foci at the poles, the mid-cell positions, and between the nucleoids at regular intervals. Calcium-dependent physical interaction between S100B and FtsZ was demonstrated in vitro by affinity chromatography, and this interaction was severely inhibited by the competitor peptide TRTK-12. Together these results indicate that S100B interacts with the tubulin homologue FtsZ in vivo, modulating its activity in bacterial cell division. This approach will present an important step for the study of S100 protein interactions in vivo.

Bacterial Proteins↗
PubMed2003

Unmasking the annexin I interaction from the structure of Apo-S100A11.

S100A11 is a homodimeric EF-hand calcium binding protein that undergoes a calcium-induced conformational change and interacts with the phospholipid binding protein annexin I to coordinate membrane association. In this work, the solution structure of apo-S100A11 has been determined by NMR spectroscopy to uncover the details of its calcium-induced structural change. Apo-S100A11 forms a tight globular structure having a near antiparallel orientation of helices III and IV in calcium binding site II. Further, helices I and IV, and I and I', form a more closed arrangement than observed in other apo-S100 proteins. This helix arrangement in apo-S100A11 partially buries residues in helices I (P3, E11, A15), III (V55, R58, M59), and IV (A86, C87, S90) and the linker (A45, F46), which are required for interaction with annexin I in the calcium-bound state. In apo-S100A11, this results in a "masked" binding surface that prevents annexin I binding but is uncovered upon calcium binding.

Annexin A1↗
PubMed2003

Interaction of the tail with the catalytic region of a class II E2 conjugating enzyme.

Ubiquitination plays an important role in many biological processes, including DNA repair, cell cycle regulation, and protein degradation. In the latter pathway the ubiquitin-conjugating enzymes or E2 enzymes are important proteins forming a key E2-ubiquitin thiolester prior to substrate labelling. While the structure of the 150-residue catalytic domain has been well characterized, a subset of E2 enzymes (class II) carry a variable length C-terminal "tail" where structural detail is not available. The presence of this C-terminal extension plays an important role in target recognition, ubiquitin chain assembly and oligomerization. In this work NMR spectroscopy was used to determine the secondary structure of the 215-residue yeast E2 protein Ubc1 and the interactions of its C-terminus with the catalytic domain. The C-terminal tail of Ubc1 was found to contain three alpha-helices between residues D169-S176, K183-L193 and N203-L213 providing the first evidence for a well-defined secondary structure in this region. Chemical shift mapping indicated that residues in the L2 loop of the catalytic domain were most affected indicating the C-terminus of Ubc1 likely interacts with this region. This site of interaction is distinct from that observed in the E2-ubiquitin thiolester and may act to protect the catalytic C88 residue and direct the interaction of ubiquitin in the thiolester intermediate.

Binding Sites↗
PubMed2002

A novel S100 target conformation is revealed by the solution structure of the Ca2+-S100B-TRTK-12 complex.

The Alzheimer-linked neural protein S100B is a signaling molecule shown to control the assembly of intermediate filament proteins in a calcium-sensitive manner. Upon binding calcium, a conformational change occurs in S100B exposing a hydrophobic surface for target protein interactions. The synthetic peptide TRTK-12 (TRTKIDWNKILS), derived from random bacteriophage library screening, bears sequence similarity to several intermediate filament proteins and has the highest calcium-dependent affinity of any target molecule for S100B to date (K(d) <1 microm). In this work, the three-dimensional structure of the Ca(2+)-S100B-TRTK-12 complex has been determined by NMR spectroscopy. The structure reveals an extended, contiguous hydrophobic surface is formed on Ca(2+)-S100B for target interaction. The TRTK-12 peptide adopts a coiled structure that fits into a portion of this surface, anchored at Trp(7), and interacts with multiple hydrophobic contacts in helices III and IV of Ca(2+)-S100B. This interaction is strikingly different from the alpha-helical structures found for other S100 target peptides. By using the TRTK-12 interaction as a guide, in combination with other available S100 target structures, a recognition site on helix I is identified that may act in concert with the TRTK-12-binding site from helices III and IV. This would provide a larger, more complex site to interact with full-length target proteins and would account for the promiscuity observed for S100B target protein interactions.

Amino Acid Sequence↗
PubMed2002

The C-terminus and linker region of S100B exert dual control on protein-protein interactions with TRTK-12.

S100B, an EF-hand calcium-binding protein composed of two S100beta monomers, undergoes a calcium-dependent conformational change that provides a surface for target interactions. In this study, the calcium-sensitive S100B-binding epitope TRTK-12 has been used to probe the contributions of the linker and C-terminal regions of S100B to protein-protein interactions. These contributions were quantified using C-terminal mutant S100B proteins lacking the C-terminal seven (S100B85stop) or nine (S100B83stop) residues or containing alanine substitutions at Phe87 (F87A), Phe88 (F88A), or both (F8788A). Both F8788A and F88A bound TRTK-12 less tightly (K(d) = 1.85 +/- 0.02 and 0.97 +/- 0.08 microM, respectively) than the wild-type protein (K(d) = 0.27 +/- 0.03 microM, DeltaG = -37.2 kJ/mol), indicating these residues are important for TRTK-12 interaction. The truncated S100B proteins bound TRTK-12 much more weakly (K(d) = 659.7 +/- 119.3 microM, DeltaG = -17.9 kJ/mol), indicating the linker region contributed about 50% to the binding of TRTK-12, while the C-terminus contributed the remaining 50% of the binding energy. Based on mutagenesis and NMR chemical shift studies, a comparison with known S100-target protein complexes showed the S100B-TRTK-12 complex has the strongest resemblance to the S100A10-annexin II interaction.

Amino Acid Sequence↗
PubMed2002

Role of the N-terminal helix I for dimerization and stability of the calcium-binding protein S100B.

Human S100B(beta beta) is a small intracellular EF-hand calcium-binding protein that consists of two noncovalently associated 91-residue beta monomers. The three-dimensional structures of S100B reveal the dimer interface consists of four alpha-helices (I, I' and IV, IV') packed in an X-type bundle. In this study, guanidine hydrochloride denaturation and dynamic light scattering were used to assess the impact of single (L3A, L3S, M7A, I11A, F14A) and double (L3A/I11A and L3A/F14A) substitution mutations in helix I on the stability and dimerization propensity of S100B. The free energy of unfolding (Delta G(u)) of wild-type apo-S100B was determined to be 72.4 +/- 4.0 kJ mol(-1), consistent with it being the most stable calcium-binding protein to date. The order of stability of the mutants in their apo form is S100B > L3A > L3S > I11A > M7A approximately L3A/I11A > F14A > L3A/F14A. Further, there is a strong correlation between the stability and the cooperativity of unfolding. Each mutation proved to be more stable in its calcium form compared to its apo form. The calcium-bound L3S substitution proved to be significantly more stable than calcium-saturated S100B, whereas the L3A, I11A, and L3A/I11A mutants are only slightly more stable than the wild-type protein. The F14A and L3A/F14A mutants are significantly reduced in stability, even in the presence of calcium.

Base Sequence↗
PubMed2002

1H, 15N and 13C resonance assignments of rabbit apo-S100A11.

S100 proteins belong to the EF-hand family of calcium binding proteins. Upon calcium binding, these proteins undergo a conformational change to expose a hydrophobic region necessary for target protein interaction. One member of the S100 protein family is S100A11, first isolated from chicken gizzard and termed calgizzarin. It was later isolated from other organisms and tissues including human placenta, pig heart and rabbit lung. The physiological target of S100A11 is thought to be annexin I, a phospholipid-binding protein involved in EGF receptor sorting. This work reports the 1H, 15N and 13C resonance assignments of rabbit apo-S100A11 determined using 15N, 13C-labelled protein and multidimensional NMR spectroscopy.

Animals↗
PubMed2002

Folding and stability of the b subunit of the F(1)F(0) ATP synthase.

The F(1)F(0) ATP synthase is a reversible molecular motor that employs a rotary catalytic cycle to couple a chemiosmotic membrane potential to the formation/hydrolysis of ATP. The multisubunit enzyme contains two copies of the b subunit that form a homodimer as part of a narrow, peripheral stalk structure that connects the membrane (F(0)) and soluble (F(1)) sectors. The three-dimensional structure of the b subunit is unknown making the nature of any interactions or conformational changes within the F(1)F(0) complex difficult to interpret. We have used circular dichroism and analytical ultracentrifugation analyses of a series of N- and C-terminal truncated b proteins to investigate its stability and structure. Thermal denaturation of the b constructs exhibited distinct two-state, cooperative unfolding with T(m) values between 30 and 40 degrees C. CD spectra for the region comprising residues 53-122 (b(53-122)) showed theta;(222)/theta;(208) = 0.99, which reduced to 0.92 in the presence of the hydrophobic solvent trifluoroethanol. Thermodynamic parameters for b(53-122) (DeltaG, DeltaH and DeltaC(p)) were similar to those reported for several nonideal, coiled-coil proteins. Together these results are most consistent with a noncanonical and unstable parallel coiled-coil at the interface of the b dimer.

Amino Acid Sequence↗