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Structural determinants for the glucose transport kinetics of the erythrocyte glucose transporter have not been established. In this work the role of the cytosolic carboxy-terminal tail in the expression and function of the human GLUT1 isoform in Xenopus oocytes was investigated. Oocyte plasma membrane expression of GLUT1 was a saturable function of the amount of mRNA injected. Transport activity increased as a linear function of the amount of immunoreactive transporter in the plasma membrane. Transport kinetics of human GLUT1 expressed in oocytes resembled those of human erythrocyte GLUT1. Addition of up to 31 extra amino acids to the carboxy-terminal tail of GLUT1 was without effect on its function in oocytes. Removal of the carboxy-terminal 21 amino acids also did not affect GLUT1 expression or transport kinetics in oocytes. Removal of the entire carboxy-terminal tail to Phe-450 resulted in a transporter that had moderately decreased plasma membrane expression compared to that of GLUT1. However, transport activity of this construct was less than 5% of that of GLUT1, and was associated with loss of its outward-facing inhibitor binding site. When the carboxy-terminal 29 amino acids of GLUT1 were replaced with the corresponding region of GLUT4, transporter expression in the plasma membrane and the transport Vmax fell to low levels, similar to those of native GLUT4. When the carboxy-terminal 29 or 73 amino acids of GLUT1 were swapped into the corresponding region of GLUT4, the transport Vmax markedly increased to about one-third to one-half that of GLUT1, although the affinity for substrate was halved. These results show that the carboxy-terminal tail of the GLUT1 is not critical for targeting of the protein to the plasma membrane, but that this region is an important determinant of transport function.
Protein tyrosine phosphatases (PTPs) are important enzymes involved in signal transduction, cell cycle regulation, and the control of differentiation. Despite the importance of this class of enzymes in the control of critical cell processes, very little structural information is available for this family of proteins. In this paper, we present the first solution structure of a protein tyrosine phosphatase. This protein is a low molecular weight cytosolic PTP that was initially isolated from bovine heart. The structure that was determined from 1747 NMR-derived restraints consists of a central four-stranded parallel beta-sheet surrounded by four alpha-helices and a short 3(10) helix. The phosphate binding site, identified by chemical shift changes upon the addition of the competitive inhibitors phosphate and vanadate, is in a loop region connecting the C-terminal end of the first beta-strand with the first alpha-helix. Residues in the second, fourth, and fifth alpha-helices and in some of the loop regions connecting the elements of regular secondary structure also contribute to the binding site. The structure determined here is consistent with previous mutagenesis and chemical modification studies conducted on this protein.
The 1H, 13C, and 15N resonances of FKBP when bound to the immunosuppressant, ascomycin, were assigned using a computer-aided analysis of heteronuclear double and triple resonance three-dimensional nmr spectra of [U-15N]FKBP/ascomycin and [U-15N,13C]FKBP/ascomycin. In addition, from a preliminary analysis of two heteronuclear four-dimensional data sets, 3JHN,H alpha coupling constants, amide exchange data, and the differences between the C alpha and C beta chemical shifts of FKBP to random coil values, the secondary structure of FKBP when bound to ascomycin was determined. The secondary structure of FKBP when bound to ascomycin in solution closely resembled the x-ray structure of the FKBP/FK506 complex but differed in some aspects from the structure of uncomplexed FKBP in solution.
A high-resolution three-dimensional solution structure of the FKBP/ascomycin complex has been determined using heteronuclear multidimensional nuclear magnetic resonance spectroscopy (NMR) and a distance geometry/simulated annealing protocol. A total of 43 structures of the complex, including 3 tightly bound water molecules, were obtained using 1958 experimental restraints consisting of 1724 nuclear Overhauser effect (NOE) derived distances, 66 chi 1 and 46 phi angular restraints, and 122 hydrogen bond restraints. The root mean square (rms) deviations between the 43 FKBP/ascomycin solution structures and the mean atomic coordinates were 0.43 +/- 0.08 A for the backbone heavy atoms and 0.80 +/- 0.08 A for all non-hydrogen atoms. Angular order parameters for the family of 43 conformations were calculated to determine dihedral convergence. Order parameters for phi, psi, and chi 1 angles exhibited mean values of 0.98, 0.97, and 0.95, respectively, while the mean of the chi 2 order parameter was 0.63. Comparisons were made between the FKBP/ascomycin complex and two NMR-derived solution structures of unbound FKBP and the X-ray crystal structure of an FKBP/FK506 complex. Differences were observed between the FKBP/ascomycin complex and uncomplexed FKBP for residues 33-45 and 78-92. In contrast, the NMR-derived solution structure of the FKBP/ascomycin complex and the X-ray crystal structure of the FKBP/FK506 complex were very similar. Differences between the two complexes were mainly observed in the conformations of some highly solvent exposed side chains.
The three-dimensional solution structure of (Cd2+)1-calbindin D9k has been determined by distance geometry, restrained molecular dynamics and relaxation matrix calculations using experimental constraints obtained from two-dimensional 1H and 15N-1H NMR spectroscopy. The final input data consisted of 1055 NOE distance constraints and 71 dihedral angle constraints, corresponding to 15 constraints per residue on average. The resulting ensemble of 24 structures has no distance or dihedral angle constraints consistently violated by more than 0.07 A and 1.8 degrees, respectively. The structure is characteristic of an EF-hand protein, with two helix-loop-helix calcium binding motifs joined by a flexible linker, and a short anti-parallel beta-type interaction between the two ion-binding sites. The four helices are well defined with a root mean square deviation from the mean coordinates of 0.35 A for the backbone atoms. The structure of the half-saturated cadmium state was compared with the previously determined solution structures of the apo and fully calcium saturated calbindin D9k. The comparisons were aided by introducing the ensemble averaged distance difference matrix as a tool for analyzing differences between two ensembles of structures. Detailed analyses of differences between the three states in backbone and side-chain dihedral angles, hydrogen bonds, interatomic distances, and packing of the hydrophobic core reveal the reorganization of the protein that occurs upon ion binding. Overall, it was found that (Cd2+)1-calbindin D9k, representing the half-saturated calcium state with an ion in site II, is structurally more similar to the fully calcium-saturated state than the apo state. Thus, for the binding sequence apo-->(Ca2+)II1-->(Ca2+)I,II2, the structural changes occurring upon ion binding are most pronounced for the first binding step, an observation that bears significantly on the molecular basis for cooperative calcium binding in calbindin D9k.
She is a widely expressed adapter protein that plays an important role in signaling via a variety of cell surface receptors and has been implicated in coupling the stimulation of growth factor, cytokine, and antigen receptors to the Ras signaling pathway. She interacts with several tyrosine-phosphorylated receptors through its C-terminal SH2 domain, and one of the mechanisms of T-cell receptor-mediated Ras activation involves the interaction of the Shc SH2 domain with the tyrosine-phosphorylated zeta chain of the T-cell receptor. Here we describe a high-resolution NMR structure of the Shc SH2 domain complexed to a phosphopeptide (GHDGLpYQGLSTATK) corresponding to a portion of the zeta chain of the T-cell receptor. Although the overall architecture of the protein is similar to other SH2 domains, distinct structural differences were observed in the smaller beta-sheet, BG loop, (pY + 3) phosphopeptide-binding site, and relative position of the bound phosphopeptide.
Characterizing the structure properties of unfolded proteins is important for understanding the stability and folding of native proteins. However, little structural information is available for the unfolded state. Using recently developed heteronuclear multi-dimensional NMR techniques, the 1H, 13C and 15N chemical shift assignments of the FK506 binding protein (FKBP) unfolded in concentrated urea and guanidine hydrochloride (GuHCl) solutions have been obtained, and the structural properties of unfolded FKBP have been characterized. FKBP displays extensive conformational averaging when unfolded in urea and GuHCl, but defined regions of secondary structure are present. Subtle differences regarding the location and stability of the secondary structures exist between the two solvents. Secondary structure formation in unfolded FKPB was correlated with statistical and thermodynamic predictions of helix formation as well as with the three-dimensional structure of folded FKBP determined by NMR and X-ray crystallography. Residues involved in secondary structures in unfolded FKBP are generally found in the same type of secondary structure in the folded protein. An exception to this was found at the C terminus of FKBP, which forms a different secondary structure in the unfolded and folded states.
Crystallographic analysis of 2.2 angstrom resolution shows that guanosine triphosphate (GTP) hydrolysis triggers conformational changes in the heterotrimeric G-protein alpha subunit, Gi alpha 1. The switch II and switch III segments become disordered, and linker II connecting the Ras and alpha helical domains moves, thus altering the structures of potential effector and beta gamma binding regions. Contacts between the alpha-helical and Ras domains are weakened, possibly facilitating the release of guanosine diphosphate (GDP). The amino and carboxyl termini, which contain receptor and beta gamma binding determinants, are disordered in the complex with GTP, but are organized into a compact microdomain on GDP hydrolysis. The amino terminus also forms extensive quaternary contacts with neighboring alpha subunits in the lattice, suggesting that multimers of alpha subunits or heterotrimers may play a role in signal transduction.
The interaction between fibrinogen gamma-peptide 392-411, LTIGEGQQHHLGGAKQAGDV, and monoclonal antibody 4A5, an antibody with a high affinity for both for the peptide and native fibrinogen, is being studied as a model for peptide-antibody interaction. Two-dimensional NMR studies of the free peptide at pH 5.2 indicated the presence of a significant population, about 60%, of type II beta-turn, spanning residues Gln407-Asp410. At pH 2.7, little, if any, turn structure is present. The D-Ala409 analog, which, for steric reasons, would be expected to preserve the beta-turn, and the L-Ala409 analog, which would not be expected to have this conformational feature, were synthesized, and NMR studies confirmed the respective structural predictions. The affinity of the D-Ala analog for antibody 4A5 is even greater than that displayed by native gamma 392-411, while the affinity of the L-Ala analog is less than one-tenth that of the native peptide. Both conformational and steric effects involving residues 407-410 may be important in recognition by antibody 4A5. Since gamma 392-411 includes a platelet receptor binding locus of fibrinogen, and this and related peptides are inhibitors of platelet aggregation, the D-Ala409 and L-Ala409 analogs were tested for platelet binding. Neither of the analogs displays any measurable platelet binding, indicating that the recognition requirements for the platelet receptor differ considerably from those for antibody 4A5.
We have identified a novel family of yeast nuclear pore complex proteins. Three individual members of this family, NUP49, NUP100, and NUP116, have been isolated and then characterized by a combination of molecular genetics and immunolocalization. Employing immunoelectron and immunofluorescence microscopy on yeast cells, we found that the binding of a polyspecific monoclonal antibody recognizing this family was predominantly at the nuclear pore complexes. Furthermore, the tagging of NUP49 with a unique epitope enabled the immunolocalization of this protein to the nuclear pore complex by both fluorescence and electron microscopy. DNA sequence analysis has shown that the amino-terminal regions of NUP49, NUP100, and NUP116 share repeated "GLFG" motifs separated from each other by glutamine, asparagine, serine and threonine rich spacers. All three proteins lack a repetitive domain found in the two precisely described yeast nuclear pore complex proteins. Only NUP49 is essential for cell viability. NUP116-deficient cells grow very slowly and are temperature sensitive, whereas the lack of NUP100 has no detectable phenotype. NUP100 and NUP116 are homologous over their entire lengths. Interestingly, NUP100 and NUP116 are both flanked by a histidine tRNA gene and a transposon element suggesting that they may have arisen by gene duplication. We propose that subfamilies of pore complex proteins can be defined by their characteristic combinations of different modular domains.