共查询到20条相似文献,搜索用时 31 毫秒
1.
JW Crabb A Carlson Y Chen S Goldflam R Intres KA West JD Hulmes JT Kapron LA Luck J Horwitz D Bok 《Canadian Metallurgical Quarterly》1998,7(3):746-757
Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Müller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration. The protein carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autosomal recessive retinitis pigmentosa. CRALBP is also present in brain without endogenous retinoids, suggesting other ligands and physiological roles exist for the protein. Human recombinant cellular retinaldehyde-binding protein (rCRALBP) has been over expressed as non-fusion and fusion proteins in Escherichia coli from pET3a and pET19b vectors, respectively. The recombinant proteins typically constitute 15-20% of the soluble bacterial lysate protein and after purification, yield about 3-8 mg per liter of bacterial culture. Liquid chromatography electrospray mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structure and mass. Circular dichroism, retinoid HPLC, UV-visible absorption spectroscopy, and solution state 19F-NMR were used to characterize the secondary structure and retinoid binding properties of rCRALBP. Human rCRALBP appears virtually identical to bovine retinal CRALBP in terms of secondary structure, thermal stability, and stereoselective retinoid-binding properties. Ligand-dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exhibited by bovine and human CRALBP when complexed with 9-cis-retinal. These recombinant preparations provide valid models for human CRALBP structure-function studies. 相似文献
2.
While the overall biosynthetic pathway leading from all-trans-retinoids to 11-cis-retinoids in the visual cycle is understood, little is known about which step(s) may be rate-limiting and how control is exerted. One possible target for control is the isomerohydrolase, which processes all-trans-retinyl esters into 11-cis-retinol. The basal rate of 11-cis-retinol synthesis from all-trans-retinyl esters is extremely slow using bovine retinal pigment epithelial membranes [3.5 pmol of 11-cis-retinol min-1 (mg of protein)-1], and only small amounts of 11-cis-retinyl ester are formed. However, the addition of retinol binding proteins stimulates 11-cis-retinol formation by a factor of approximately 13. Specific protein-protein interactions are probably unimportant because bovine serum albumin and the physiologically relevant cellular retinaldehyde binding protein (CRALBP) both stimulate 11-cis-retinol formation to the same extent, although CRALBP does so at much lower concentrations. The relatively rapid rate of isomerization in the presence of binding proteins [44.3 pmol of 11-cis-retinol min-1 (mg of protein)-1] suggests that the rate-limiting enzyme in the visual cycle need not be the isomerohydrolase. Also, 11-cis-retinol is shown to inhibit isomerohydrolase, providing a simple mechanism for regulation of the visual cycle and the stimulating effect of binding proteins. 相似文献
3.
BN Kennedy S Goldflam MA Chang P Campochiaro AA Davis DJ Zack JW Crabb 《Canadian Metallurgical Quarterly》1998,273(10):5591-5598
Cellular retinaldehyde-binding protein (CRALBP) is abundantly expressed in the retinal pigment epithelium (RPE) and Muller cells of the retina, where it is thought to function in retinoid metabolism and visual pigment regeneration. Mutations in human CRALBP that destroy retinoid binding have been linked to autosomal recessive retinitis pigmentosa. To identify the DNA elements that regulate expression of the human CRALBP gene in the RPE, transient transfection studies were carried out with three CRALBP-expressing human RPE cell culture systems. The regions from -2089 to -1539 base pairs and from -243 to +80 base pairs demonstrated positive regulatory activity. Similar activity was not observed with cultured human breast, liver, or skin cells. Since sequence analysis of the -243 to +80 region identified the presence of two photoreceptor consensus element-1 (PCE-1) sites, elements that have been implicated in photoreceptor gene regulation, the role of these sequences in RPE expression was examined. Mutation of either PCE-1 site significantly reduced reporter activity, and mutation or deletion of both sites dramatically reduced activity. Electrophoretic mobility shift analysis with RPE nuclear extracts revealed two complexes that required intact PCE-1 sites. These studies also identified two identical sequences (GCAGGA) flanking PCE-1, termed the binding CRALBP element (BCE), that are also important for complex formation. Southwestern analysis with PCE-1/BCEcontaining probes identified species with apparent masses near 90-100 and 31 kDa. These results begin to identify the regulatory regions required for RPE expression of CRALBP and suggest that PCE-1-binding factor(s) may play a role in regulating RPE as well as photoreceptor gene expression. 相似文献
4.
JA Fernández JH Griffin GT Chang J Stam PH Reitsma RM Bertina BN Bouma 《Canadian Metallurgical Quarterly》1998,24(2):101-12; discussion 113
Human protein S binds to C4b-binding protein (C4BP) both in plasma and in a system using purified proteins. Amino acid residues 420-434 of the first disulfide loop of the sex hormone binding globulinlike domain of protein S are involved in the interaction of protein S with C4BP. To define the involvement of specific polar amino acids within residues 420-434, we studied in parallel synthetic protein S peptides and recombinant protein S variants containing the same amino acid replacements, K423E, E424K, Q427E and K429E. Synthetic peptide analogs of peptide PSP-420 (residues 420-434) were assayed for binding C4BP and as inhibitors of complex formation. The PSP-420 peptide and the analogous peptide with the substitution E424K, but not the peptides containing the substitutions K423E and K429E, were able to bind C4BP. Recombinant proteins with mutations of K423E, Q427E and K429E showed reduced affinity for C4BP compared to plasma protein S, recombinant wild type protein S, or E424K-protein S. These results suggest that Lys-423, Gln-427 and Lys-429 of protein S are important for normal binding to C4BP. The anti-protein S monoclonal antibody LJ-56, raised against peptide PSP-420, recognizes only free protein S and inhibits complex formation with C4BP. Antibody LJ-56 recognized the E424K and Q427E peptides but not the K423E or K429E peptides. Similarly, the E424K and Q427E protein S mutants were recognized by LJ-56, whereas the K423E and K429E protein S mutants were not recognized. This suggests that both in the peptide PSP-420 and in protein S, Lys-423 and Lys-429 significantly contribute to binding to antibody LJ-56. These results demonstrate that protein S residues 423, 427 and 429, but not residue 424, are involved in binding to both the antibody LJ-56 and to C4BP. When peptides PSP 420 and SL-6 (residues 447-460) with carboxyterminal amide or carboxylate moieties were compared to their ability to inhibit C4BP-protein S complexation, PSP-420-amide was the most potent. This finding together with the other results described here supports the hypothesis that the residues 420 and 434 in protein S provides a major binding site for C4BP. 相似文献
5.
RPE65 is a major membrane associated protein found in the vertebrate retinal pigment epithelium (RPE). Various studies have shown this protein to be essential for visual function, possibly at the level of the processing of retinoids. The pigment epithelium is the anatomical site in which the visual chromophore 11-cis retinal is generated. The two critical RPE enzymes in the isomerization pathway are lecithin retinol acyl transferase (LRAT) and isomerohydrolase, which processes all-trans-retinyl esters into 11-cis-retinol. Both enzymes are membrane bound. It is shown here that RPE65 can be largely extracted (90-95%) from RPE membranes by 1 M KCl by itself, or with added detergent CHAPS. The almost quantitative extraction of RPE65 from RPE membranes has little or no effect on in vitro LRAT and isomerohydrolase activities in quantitative enzymatic assays using RPE membranes, suggesting that RPE65 may not have an important role to play in the enzymatic processing of all-trans-retinol into 11-cis-retinol in vitro. 相似文献
6.
W Shi CD Sports D Raman S Shirakawa S Osawa ER Weiss 《Canadian Metallurgical Quarterly》1998,37(14):4869-4874
Arginine-135, located at the border between the third transmembrane domain and the second cytoplasmic loop of rhodopsin, is one of the most highly conserved amino acids in the family of G protein-coupled receptors. The effect of mutation at Arg-135 on the ability of rhodopsin to undergo desensitization was investigated. Four mutants, R135K, R135Q, R135A, and R135L, were examined for their ability to be phosphorylated by rhodopsin kinase, to bind arrestin, and to activate the rod cell G protein, transducin (Gt). All of the mutants were phosphorylated, bound arrestin, and were able to activate Gt when reconstituted with 11-cis-retinal. Surprisingly, several of the mutants could be phosphorylated by rhodopsin kinase and could bind arrestin in the absence of 11-cis-retinal but were not able to activate Gt. These observations represent the first demonstration of a mutant G protein-coupled receptor that assumes a conformation able to interact with its G protein-coupled receptor kinase and arrestin, but not with its G protein, in the absence of ligand. 相似文献
7.
Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium and Müller glial cells of the retina, where it forms complexes with endogenous 11-cis-retinoids. We examined the distribution of CRALBP in extraretinal tissues using polyclonal antibodies (pAb) and monoclonal antibodies (mAb). A protein was detected by immunoblot analysis in extracts of bovine and rat brain and optic nerve but not in several other tissues. This protein had electrophoretic, chromatographic, and retinoid-binding properties identical to those of CRALBP from bovine retina. Comparison of the masses of tryptic peptides and of partial amino acid sequences derived from brain and retinal CRALBP indicated that the two proteins are probably identical. Immunoperoxidase cytochemistry and double labeling immunofluorescence revealed CRALBP(+) cells in brain that resembled oligondendrocytes and not astrocytes, microglial cells, or pinealocytes. In 11-day-old rat brain, approximately 11% of the CRALBP(+) cells were labeled with the Rip antibody, a marker for oligodendroglia. In developing rat optic nerve, the temporal appearance of CRALBP(+) cells corresponded to that of oligodendrocytes and not that of astrocytes. In adult rat and mouse optic nerves, the CRALBP(+) somata showed the same distribution as oligodendrocytes. No endogenous retinoids were associated with CRALBP isolated from dark-dissected adult bovine brain. The results suggest that CRALBP has functions in addition to retinoid metabolism and visual pigment regeneration. 相似文献
8.
RD Chessick 《Canadian Metallurgical Quarterly》1998,26(2):209-222
E. coli catalase (HPII) wild type and mutant enzymes (heme dcis-containing) were examined (i) to study the role of a distal haem cavity residue, asparagine-201, in high spin ligand binding and (ii) to compare the differences in this binding between heme d and protoheme enzymes such as that from beef liver (BLC). High spin fluoride complexes were formed by all three HPII catalases examined, wild type (201 asn) and 201gln and 201asp mutants, but with a lower fluoride affinity than that of BLC. The binding of fluoride was pH-dependent, indicating that a proton is bound as well as a fluoride anion. HPII 201glu and 201 asp mutants showed lower affinities for fluoride than did wild type, unlike their reactions with cyanide which are essentially independent of the nature of residue 201. The equilibria and rates of fluoride and formate binding to BLC were reexamined. The rates of reaction with formate were similar to those reported previously. Dissociation rates for fluoride-catalase are higher than for formate suggesting that the latter may be bound differently. High spin complexes between formate and all three HPII forms showed a substantially higher affinity than that of BLC for HPII wild type and progressively lower affinities for the two mutants. As with fluoride the reactions were pH-dependent, indicating that a proton is bound together with the formate anion (or that undissociated formic acid is the ligand). The known structures of the heme groups and heme pockets involved are discussed. Formate may be bound by secondary H-bounds within the heme pocket in both heme dcis and protoheme enzymes. The nature of the heme pocket and the heme access channel may be more important than the chemical nature of the prosthetic group in controlling both high spin ligand interactions and reactions with the substrate hydrogen peroxide. 相似文献
9.
In this study, we have examined the transport characteristics of the wild-type lactose permease, single mutants in which Lys-319 was changed to asparagine or alanine or Glu-325 was changed to glutamine or alanine, and the corresponding double mutant strains. The wild-type and Asn-319 mutant showed high levels of lactose uptake, with Km values of 0.42 and 1.30 mM and Vmax values of 102.6 and 48.3 nmol of lactose/min/mg of protein, respectively. The Asn-319/Gln-325 strain had a normal Km of 0.36 mM and a moderate Vmax of 18.5 nmol of lactose/min/mg of protein. By comparison, the single E325Q strain had a normal Km of 0.27 mM but a very defective Vmax of 1.3 nmol of lactose/min/mg of protein. A similar trend was observed among the alanine substitutions at these positions, although the Vmax values were lower for the Ala-319 mutations. When comparing the Vmax values between the single position 325 mutants with those of the double mutants, these results indicate that neutral 319 mutations substantially alleviate a defect in Vmax caused by neutral 325 mutations. With regard to H+/lactose coupling, the wild-type permease is normally coupled and can transport lactose against a gradient. The position 325 single mutants showed no evidence of H+ transport with lactose or thiodigalactoside (TDG) and were unable to facilitate uphill lactose transport. The single Asn-319 mutant and double Asn-319/Gln-325 mutant were able to transport H+ upon the addition of lactose or TDG. In addition, both of these strains catalyzed a sugar-dependent H+ leak that inhibited cell growth in the presence of TDG. These two strains were also defective in uphill transport, which may be related to their sugar-dependent leak pathway. Based on these and other results in the literature, a model is presented that describes how the interactions among several ionizable residues within the lactose permease act in a concerted manner to control H+/lactose coupling. In this model, Lys-319 and Glu-325 play a central role in governing the ability of the lactose permease to couple the transport of H+ and lactose. 相似文献
10.
FK506 binding protein mutational analysis. Defining the surface residue contributions to stability of the calcineurin co-complex 总被引:2,自引:0,他引:2
O Futer MT DeCenzo RA Aldape DJ Livingston 《Canadian Metallurgical Quarterly》1995,270(32):18935-18940
The 12- and 13-kDa FK506 binding proteins (FKBP12 and FKBP13) are cis-trans peptidyl-prolyl isomerases that bind the macrolides FK506 (Tacrolimus) and rapamycin (Sirolimus). The FKBP12.FK506 complex is immunosuppressive, acting as an inhibitor of the protein phosphatase calcineurin. We have examined the role of the key surface residues of FKBP12 and FKBP13 in calcineurin interactions by generating substitutions at these residues by site-directed mutagenesis. All mutants are active catalysts of the prolyl isomerase reaction, and bind FK506 or rapamycin with high affinity. Mutations at FKBP12 residues Asp-37, Arg-42, His-87, and Ile-90 decrease calcineurin affinity of the mutant FKBP12.FK506 complex by as much as 2600-fold in the case of I90K. Replacement of three FKBP13 surface residues (Gln-50, Ala-95, and Lys-98) with the corresponding homologous FKBP12 residues (Arg-42, His-87, and Ile-90) generates an FKBP13 variant that is equivalent to FKBP12 in its affinity for FK506, rapamycin, and calcineurin. These results confirm the role of two loop regions of FKBP12 (residues 40-44 and 84-91) as part of the effector face that interacts with calcineurin. 相似文献
11.
The Escherichia coli Fpg protein is involved in the repair of oxidized residues. We examined, by targeted mutagenesis, the effect of the conserved lysine residue at position 57 upon the various catalytic activities of the Fpg protein. Mutant Fpg protein with Lys-57-->Gly (K57G) had dramatically reduced DNA glycosylase activity for the excision of 7,8-dihydro-8-oxo-guanine (8-oxoG). While wild type Fpg protein cleaved 8-oxoG/C DNA with a specificity constant ( k cat/ K M) of 0.11/(nM@min), K57G cleaved the same DNA 55-fold less efficiently. FpgK57G was poorly effective in the formation of Schiff base complex with 8-oxoG/C DNA. The efficiency in the binding of 8-oxoG/C DNA duplex for K57G mutant was decreased 16-fold. The substitution of Lys-57 for another basic amino acid Arg (K57R) had a slight effect on the 8-oxoG-DNA glycosylase activity and Schiff base formation. The DNA glycosylase activities of FpgK57G and FpgK57R using 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine residues as substrate were comparable to that of wild type Fpg. In vivo, the mutant K57G, in contrast to the mutant K57R and wild type Fpg, only partially restored the ability to prevent spontaneously induced transitions G/C-->T/A in E.coli BH990 ( fpg mutY ) cells. These results suggest an important role for Lys-57 in the 8-oxoG-DNA glycosylase activity of the Fpg protein in vitro and in vivo. 相似文献
12.
The crystal structure of Escherichia coli ornithine transcarbamoylase (OTCase, EC 2.1.3.3) complexed with the bisubstrate analog N-(phosphonacetyl)-L-ornithine (PALO) has been determined at 2.8-A resolution. This research on the structure of a transcarbamoylase catalytic trimer with a substrate analog bound provides new insights into the linkages between substrate binding, protein-protein interactions, and conformational change. The structure was solved by molecular replacement with the Pseudomonas aeruginosa catabolic OTCase catalytic trimer (Villeret, V., Tricot, C., Stalon, V. & Dideberg, O. (1995) Proc. Natl. Acad. Sci. USA 92, 10762-10766; Protein Data Bank reference pdb 1otc) as the model and refined to a crystallographic R value of 21.3%. Each polypeptide chain folds into two domains, a carbamoyl phosphate binding domain and an L-ornithine binding domain. The bound inhibitor interacts with the side chains and/or backbone atoms of Lys-53, Ser-55, Thr-56, Arg-57, Thr-58, Arg-106, His-133, Asn-167, Asp-231, Met-236, Leu-274, Arg-319 as well as Gln-82 and Lys-86 from an adjacent chain. Comparison with the unligated P. aeruginosa catabolic OTCase structure indicates that binding of the substrate analog results in closure of the two domains of each chain. As in E. coli aspartate transcarbamoylase, the 240s loop undergoes the largest conformational change upon substrate binding. The clinical implications for human OTCase deficiency are discussed. 相似文献
13.
DR McFarlane EF McFarlane JA Barden BE Kemp 《Canadian Metallurgical Quarterly》1993,1162(1-2):187-194
The components of secondary structure of the biologically-active N-terminal domain of human parathyroid-hormone-related protein (residues 1-34) and several truncated species were examined using Fourier transform infrared (FTIR) spectroscopy. The major structural features include a segment of alpha-helix within the N-terminal segment probably extending from Glu-4 to Lys-11 with three beta-turns localized to the segments Gly-12 to Ile-15, Gln-16 to Arg-20 and His-25 to Ala-29. Some beta-sheet was detected in the full-length peptide, but not in any of the C-terminal truncated samples. These structural features were studied in the smaller peptides for the purpose of localization of the various components and with a view to describing the region likely to form the bulk of the receptor binding site. 相似文献
14.
F Gonzalez-Fernandez CA Baer E Baker TI Okajima B Wiggert MS Braiman DR Pepperberg 《Canadian Metallurgical Quarterly》1998,17(12):1150-1157
PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP), an extracellular protein believed to support the exchange of retinoids between the neural retina and retinal pigment epithelium (RPE) in the vertebrate eye, exhibits a modular, i.e., repeat, structure. The present study was undertaken to determine whether an individual module of IRBP has activity in retinoid transfer between the RPE and rod photoreceptors. METHODS: The retinoid transfer activity of a recombinant protein corresponding to the fourth module of Xenopus laevis IRBP (X4IRBP) was examined in two ways. First, X4IRBP was tested for its ability to support the regeneration of porphyropsin in detached/reattached Xenopus retina/RPE-eyecups. Following illumination and removal of native IRBP, Xenopus eyecups supplemented with 42 microM X4IRBP or (as a control) Ringer's solution were incubated in darkness and then analyzed for regenerated porphyropsin. Second, toad (Bufo marinus) RPE-eyecup preparations were used to evaluate X4IRBP's ability to promote the release of 11-cis retinal from the RPE. RESULTS: The regeneration of porphyropsin in X4IRBP-supplemented Xenopus retina/RPE-eyecups (0.45 +/- 0.04 nmol; mean +/- SEM, n = 11) exceeded that in controls (0.13 +/- 0.02 nmol, n = 11). For promoting the release of 11-cis retinal from the toad RPE, 42 microM X4IRBP was more effective than equimolar bovine serum albumin although considerably less than that of 26 microM native bovine IRBP. CONCLUSIONS: The results indicate a low but significant activity of IRBP's fourth module in reactions relevant to retinoid exchange. 相似文献
15.
YJ Seok M Sondej P Badawi MS Lewis MC Briggs H Jaffe A Peterkofsky 《Canadian Metallurgical Quarterly》1997,272(42):26511-26521
The histidine phosphocarrier protein (HPr) is an essential element in sugar transport by the bacterial phosphoenolpyruvate:sugar phosphotransferase system. Ligand fishing, using surface plasmon resonance, was used to show the binding of HPr to a nonphosphotransferase protein in extracts of Escherichia coli; the protein was subsequently identified as glycogen phosphorylase (GP). The high affinity (association constant approximately 10(8) M-1), species-specific interaction was also demonstrated in electrophoretic mobility shift experiments by polyacrylamide gel electrophoresis. Equilibrium ultracentrifugation analysis indicates that HPr allosterically regulates the oligomeric state of glycogen phosphorylase. HPr binding increases GP activity to 250% of the level in control assays. Kinetic analysis of coupled enzyme assays shows that the binding of HPr to GP causes a decrease in the Km for glycogen and an increase in the Vmax for phosphate, indicating a mixed type activation. The stimulatory effect of E. coli HPr on E. coli GP activity is species-specific, and the unphosphorylated form of HPr activates GP more than does the phosphorylated form. Replacement of specific amino acids in HPr results in reduced GP activation; HPr residues Arg-17, Lys-24, Lys-27, Lys-40, Ser-46, Gln-51, and Lys-72 were established to be important. This novel mechanism for the regulation of GP provides the first evidence directly linking E. coli HPr to the regulation of carbohydrate metabolism. 相似文献
16.
Polar residues within the transmembrane domains (TMs) of G protein-coupled receptors have been implicated to be important determinants of receptor function. We have identified mutations at two polar sites in the TM regions of the rat parathyroid hormone (PTH)/PTH-related peptide receptor, Arg-233 in TM 2 and Gln-451 in TM 7, that caused 17-200-fold reductions in the binding affinity of the agonist peptide PTH-(1-34) without affecting the binding affinity of the antagonist/partial agonist PTH-(3-34). When mutations at the TM 2 and TM 7 sites were combined, binding affinity for PTH-(1-34) was restored to nearly that of the wild type receptor. The double mutant receptors, however, were completely defective in signaling cAMP or inositol phosphate production in response to PTH-(1-34) agonist ligand. The results demonstrate that Arg-233 and Gln-451 have important roles in determining agonist binding affinity and transmembrane signaling. Furthermore, the finding that residues in TM 2 and TM 7 are functionally linked suggests that the TM domain topology of the PTH/PTH-related peptide receptor may resemble that of receptors in the rhodopsin/beta-adrenergic receptor family, for which structural and mutagenesis data suggest interactions between TMs 2 and 7. 相似文献
17.
O Wiborg C Andersen CR Knudsen BF Clark J Nyborg 《Canadian Metallurgical Quarterly》1996,271(34):20406-20411
Two residues of Escherichia coli elongation factor Tu involved in binding of aminoacyl-tRNA were identified and subjected to mutational analysis. Lys-89 and Asn-90 were each replaced by either Ala or Glu. The four single mutants were denoted K89A, K89E, N90A, and N90E, respectively. The mutants were characterized with respect to thermal and chemical stability, GTPase activity, tRNA affinity, and activity in an in vitro translation assay. Most conspicuously tRNA affinities were reduced for all mutants. The results verify our structural analysis of elongation factor Tu in complex with aminoacyl-tRNA, which suggested an important role of Lys-89 and Asn-90 in tRNA binding. Furthermore, our results indicate helix B to be an important target site for nucleotide exchange factor EF-Ts. Also the mutants His-66 to Ala and His-118 to either Ala or Glu were characterized in an in vitro translation assay. Their functional roles are discussed in relation to the structure of elongation factor Tu in complex with aminoacyl-tRNA. 相似文献
18.
19.
T Fujisawa T Uruga Z Yamaizumi Y Inoko S Nishimura T Ueki 《Canadian Metallurgical Quarterly》1994,115(5):875-880
The small-angle X-ray scattering technique was used to characterize the structure in solution of wild type ras p21 as well as the oncogenic proteins mutated at residue 12, 59, or 61. In the presence of GDP, the radius of gyration, Rg, determined for wild type ras p21 was 16.89 +/- 0.01 A, while the wild type ras p21 bound to the GTP analogue GDPNHP (5'-guanyl imido diphosphate beta-gamma-imidoguanosine 5'-triphosphate) showed an Rg value of 17.46 +/- 0.01 A, which is 3.3% larger. The result shows that ras p21 expands upon GTP binding. The Rgs of mutated proteins were 17.04 +/- 0.01, 16.98 +/- 0.01, and 17.03 +/- 0.01 A for the Gly-12 to Val, Ala-59 to Thr, and Gln-61 to Leu mutants, respectively. The scattering profiles were analyzed by simulation of hydrated ras p21, based on the crystal atomic coordinates, and it was concluded that the ras p21 molecule incorporates 20% more bulk water upon GTP binding. The increase of bulk water is especially conspicuous around the interface between switch I (residues 32-40) and switch II (residues 60-66) regions. This suggests that hydration plays an important role in the interaction with GAP. 相似文献
20.
BACKGROUND: The integrin family of cell-surface receptors mediates a wide variety of cell-cell and cell-extracellular matrix interactions. Integrin-ligand interactions are invariably dependent on the presence of divalent cations, and a subset of integrins contain a approximately 200 amino acid inserted (I) domain that is important for ligand binding activity and contains a single divalent cation binding site. Many integrins are believed to respond to stimuli by undergoing a conformational change that increases their affinity for ligand, and there is a clear difference between two crystal structures of the CD11b I domain with different divalent cations (magnesium and manganese) bound. In addition to the different bound cation, a 'ligand mimetic' crystal lattice interaction in the CD11b I domain structure with bound magnesium has led to the interpretation that the different CD11b I domain structures represent different affinity states of I domains. The influence of the bound cation on I domain structure and function remains incompletely understood, however. The crystal structure of the CD11a I domain bound to manganese is known. We therefore set out to determine whether this structure changes when the metal ion is altered or removed. RESULTS: We report here the crystal structures of the CD11a I domain determined in the absence of bound metal ion and with bound magnesium ion. No major structural rearrangements are observed in the metal-binding site of the CD11a I domain in the absence or presence of bound manganese ion. The structures of the CD11a I domain with magnesium or manganese bound are extremely similar. CONCLUSIONS: The conformation of the CD11a I domain is not altered by changes in metal ion binding. The cation-dependence of ligand binding thus indicates that the metal ion is either involved in direct interaction with ligand or required to promote a favorable quaternary arrangement of the integrin. 相似文献