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1.
The interaction of calmodulin with fluorescent and photoreactive model peptides: evidence for a short interdomain separation 总被引:2,自引:0,他引:2
Calmodulin is known to bind target enzymes and basic, amphiphilic peptides in a Ca2(+)-dependent manner. Recently, we introduced a photoaffinity label, p-benzoylphenylalanine (Bpa), into the sequence of a model, alpha-helical, calmodulin-binding peptide. When the Bpa residue was introduced at the third position of the peptide, Met-144 on the C-terminal domain of calmodulin was labeled, whereas when the photolabel was placed at the thirteenth position, Met-71 on the N-terminal domain was labeled. Assuming that both peptides bind in similar orientations, these results are not consistent with the crystal structure of calmodulin, in which the domains are held at a significant distance from one another by a long alpha-helical segment. To test the assumption that both peptides bind in similar orientations, we have synthesized a calmodulin-binding peptide with the photolabel in both the third and the thirteenth positions. Upon photolysis, this peptide forms a cross-link between Met-71 and Met-124 on the N- and C-terminal domains, respectively. Furthermore, a peptide with a Bpa in the thirteenth position and a Trp residue in the third position was also synthesized. After photocross-linking the Bpa residue of this peptide to Met-71 of calmodulin, it could be shown that the fluorescence properties of the Trp residue were consistent with its side chain being buried in a hydrophobic pocket on the C-terminal domain of calmodulin. These data indicate that, when complexed with basic, amphiphilic peptides, calmodulin can adopt a conformation in which its two domains are significantly closer than in the crystal structure of the uncomplexed protein. 相似文献
2.
The mammalian hepatic asialoglycoprotein receptor (ASGP-R) is an endocytic recycling receptor that mediates the internalization of desialylated glycoproteins and their delivery to lysosomes where they are degraded. The human ASGP-R is a hetero-oligomeric complex composed of two subunits designated H1 and H2. Both subunits are palmitoylated at the cytoplasmic Cys residues near their transmembrane domains (TMD). The cytoplasmic Cys(36) in H1 is located at a position that is five amino acids from the transmembrane junction. Because the sequences of subunits in all mammalian ASGP-R species are highly conserved especially at the region near the palmitoylated Cys, we sought to identify a recognition signal for the palmitoylation of H1. Various types of H1 mutants were created by site-directed or deletion mutagenesis including alteration of the amino acids surrounding Cys(36), replacing portions of the TMD with that of a different protein and partial deletion of the cytoplasmic domain as well as transposing the palmitoylated Cys to positions further away from the TMD. Mutant H1 cDNAs were transiently expressed in COS-7 cells, and the H1 proteins were analyzed after metabolic labeling with [(3)H]palmitate. The results indicate that neither the native amino acid sequence surrounding Cys(36) nor the majority of the cytoplasmic domain sequence is critical for palmitoylation. Palmitoylation was also not dependent on the native TMD of H1. In contrast, the attachment of palmitate was abolished if the Cys residue was transposed to a position that was 30 amino acids away from the transmembrane border. We conclude that the spacing of a Cys residue relative to the TMD in the primary protein sequence of H1 is the major determinant for successful palmitoylation. 相似文献
3.
A large variety of proOmpF-Lpps, hybrid secretory proteins composed of the signal region of proOmpF and the mature part of the major lipoprotein, either possessing or not possessing a proline residue near the amino terminus of their mature domains, were constructed at a DNA level, and the rates of their in vitro translocation were determined in the presence and absence of the proton motive force (delta muH+). A proline residue at the signal peptide cleavage site (position +1) blocked the cleavage reaction but not the translocation reaction. All the proOmpF-Lpps examined exhibited approximately the same translocation rate in the presence of delta muH+ irrespective of the presence or absence of a proline residue near the amino terminus. In the absence of the delta muH+, which was achieved by either depletion of the respiratory substrate or the use of urea-treated membrane vesicles permeable to protons, proOmpF-Lpps possessing a proline residue near the amino terminus of the mature domain were translocated whereas those possessing no proline residue in this region were not translocated at all or only very weakly. The position of the proline residue was then moved stepwise away from the amino terminus of the mature domain. The further the position was moved away, the slower was the rate of translocation in the absence of delta muH+. The removal of the proline residue at position +2 of the mature domain of proOmpA also made the delta mu(H+)-independent translocation appreciably slower. It is suggested that the conformational flexibility endowed by the proline residue on the junction region between the signal peptide and the mature domain allows the translocation in the absence of delta muH+ and that this junction region must take on a particular conformation for initiation of the translocation reaction. 相似文献
4.
The actin-activated Mg2+-ATPase activities of Acanthamoeba myosins IA, IB, and IC are expressed only when a single site in their heavy chains is phosphorylated by a myosin I heavy chain-specific kinase. We show that phosphorylation occurs at Ser-315 in the myosin IB heavy chain, Ser-311 in myosin IC, and a threonine residue at a corresponding position in myosin IA whose amino acid sequence is as yet unknown. The most obvious feature common to the three substrates is a basic amino acid(s) 2 or 3 residues before the site of phosphorylation. The phosphorylation site is located between the ATP- and actin-binding sites, which corresponds to the middle of the 50-kDa domain of skeletal muscle myosin subfragment 1. The sequence similarity between the region surrounding the phosphorylation site of myosin I and subfragment 1 is much lower than the average sequence similarity between myosin I and subfragment 1. This is consistent with the hypothesis that the conformation of this region of myosin I differs from that of the corresponding region in skeletal muscle myosin and that phosphorylation converts the conformation of the actomyosin I complex into a conformation comparable to that present in actosubfragment 1 without phosphorylation. The protein sequences obtained in the course of this work led to the conclusion that the myosin I genes previously identified as myosin IB and IL (myosin-like) heavy chains actually are the myosin IC and IB heavy chains, respectively. Finally, we report a modification of the method for monitoring the appearance of 32Pi during sequencing of 32P-labeled peptides that results in almost complete recovery of the radioactivity, thus allowing unequivocal assignment of the position of the phosphorylated residue. 相似文献
5.
Modeling of agonist binding to the ligand-gated ion channel superfamily of receptors 总被引:2,自引:0,他引:2
A generalized model is presented of agonist binding to ligand-gated ion channels (LGICs). Broad similarity in the structure of agonists suggests that the binding sites of LGICs may have evolved from a protobinding site. Aligned sequence data identified as a candidate for such a site a highly conserved 15 residue stretch of primary structure in the N-terminal extracellular region of all known LGIC subunits. We modeled this subregion, termed the cys-loop, as a rigid, amphiphilic beta-hairpin and propose that it may form a major determinant of a conserved structural binding cleft. In the model of the binding complex (1) an invariant aspartate residue at position 11 of the cys-loop is the anionic site interacting with the positively charged amine group of agonists, (2) a local dipole within the pi-electron system of agonists is favorably oriented in the electrostatic field of the invariant aspartate, (3) the epsilon ring-proton of a conserved aromatic residue at the turn of the cys-loop interacts orthogonally with the agonist pi-electron density at its electronegative center, and (4) selective recognition is partly a result of the type of amino acid residue at position 6 of the cys-loop. Additionally, formation of a hydrogen bond between the electronegative atom of the pi-electron system of agonist and a complementary group in the receptor may be important in the high-affinity binding of agonists. 相似文献
6.
Nucleotide sequence of channel catfish heavy chain cDNA and genomic blot analyses. Implications for the phylogeny of Ig heavy chains 总被引:6,自引:0,他引:6
Our prior analyses defined the cDNA sequence on part of the CH2 domain, the complete CH3 and CH4 domains, and the 3'-untranslated region of a catfish H chain. To complete the catfish H chain mRNA sequence, a primer-extended H chain cDNA library was constructed. Analysis of this library has resulted in the definition of full-length clones encoding a 61-bp 5' untranslated region, a 51-bp leader sequence, the V region and the complete CH1 and CH2 domains. The high similarity defined with other vertebrate V regions readily allowed the catfish sequence to be divided into FR and CDR regions. Sequence comparisons with mammalian VH and JH genes strongly suggest that the catfish V region is the product of multiple genes. Using a catfish VH cDNA probe, at least 25 different genomic VH members were defined. Because this probe does not hybridize with other full-length H chain cDNA clones, additional VH families will likely be defined in catfish. Phylogenetic sequence comparisons of the catfish C region domains indicated that the CH1 and CH4 were the most highly conserved. In addition several important features were defined in genomic Southern blot analyses of catfish DNA. Gene titration experiments established that the catfish CH gene is represented by a single genomic copy. This finding provides clear evidence that the genomic organization of H chain genes in catfish must be different from that defined in sharks and suggests that the phylogeny of single copy CH genes may have been established at the level of the bony fishes. It is also likely that there is an additional CH gene in catfish. This gene is also represented by a single genomic copy, and based upon its relative signal intensity when compared with the known CH gene it appears to share higher similarity with the known CH1 domain than it does with the CH2 domain. 相似文献
7.
James Dunbar Bernhard Knapp Angelika Fuchs Jiye Shi Charlotte M. Deane 《PLoS computational biology》2014,10(9)
The variable domains of antibodies and T-Cell receptors (TCRs) share similar structures. Both molecules act as sensors for the immune system but recognise their respective antigens in different ways. Antibodies bind to a diverse set of antigenic shapes whilst TCRs only recognise linear peptides presented by a major histocompatibility complex (MHC). The antigen specificity and affinity of both receptors is determined primarily by the sequence and structure of their complementarity determining regions (CDRs). In antibodies the binding site is also known to be affected by the relative orientation of the variable domains, VH and VL. Here, the corresponding property for TCRs, the Vβ-Vα orientation, is investigated and compared with that of antibodies. We find that TCR and antibody orientations are distinct. General antibody orientations are found to be incompatible with binding to the MHC in a canonical TCR-like mode. Finally, factors that cause the orientation of TCRs and antibodies to be different are investigated. Packing of the long Vα CDR3 in the domain-domain interface is found to be influential. In antibodies, a similar packing affect can be achieved using a bulky residue at IMGT position 50 on the VH domain. Along with IMGT VH 50, other positions are identified that may help to promote a TCR-like orientation in antibodies. These positions should provide useful considerations in the engineering of therapeutic TCR-like antibodies. 相似文献
8.
Structural repertoire of the human VH segments. 总被引:16,自引:0,他引:16
C Chothia A M Lesk E Gherardi I M Tomlinson G Walter J D Marks M B Llewelyn G Winter 《Journal of molecular biology》1992,227(3):799-817
The VH gene segments produce the part of the VH domains of antibodies that contains the first two hypervariable regions. The sequences of 83 human VH segments with open reading frames, from several individuals, are currently known. It has been shown that these sequences are likely to form a high proportion of the total human repertoire and that an individual's gene repertoire produces about 50 VH segments with different protein sequences. In this paper we present a structural analysis of the amino acid sequences produced by the 83 segments. Particular residue patterns in the sequences of V domains imply particular main-chain conformations, canonical structures, for the hypervariable regions. We show that, in almost all cases, the residue patterns in the VH segments imply that the first hypervariable regions have one of three different canonical structures and that the second hypervariable regions have one of five different canonical structures. The different observed combinations of the canonical structures in the first and second regions means that almost all sequences have one of seven main-chain folds. We describe, in outline, structures of the antigen binding site loops produced by nearly all the VH segments. The exact specificity of the loops is produced by (1) sequence differences in their surface residues, particularly at sites near the centre of the combining site, and (2) sequence differences in the hypervariable and framework regions that modulate the relative positions of the loops. 相似文献
9.
A B Hartman C P Mallett S Sheriff S J Smith-Gill 《Journal of immunology (Baltimore, Md. : 1950)》1988,141(3):932-936
Nucleotide sequences of HyHEL-5, an antibody specific for chicken lysozyme (HEL), indicated unusual joins in the third complementarity-determining region of both the H and L chains. The VK-JK recombination site is unusual in that codon 96, normally derived from the JK gene segment, is deleted entirely, making the L3 one amino acid shorter than normal. Examination of the HyHEL-5 Fab-HEL x-ray structure suggests that the conformation of L3 is clearly important for Ag specificity. A comparison of the HyHEL-5 L3 with that of the structurally related antibody J539 indicates that the deleted residue significantly alters the conformation of the L3 turn. The H chain VH-DH join is also unusual; the VH junction site has probably occurred between the second and third nucleotides of codon 92, with the addition of five random nucleotides that encode for unusual amino acids Leu93 and His94. Although the conformation of H3 is different from what would be predicted from other H3 conformations and is clearly important to the complementarity of HyHEL-5 to HEL, the specific residues at the VH-DH join do not appear to directly contribute to Ag binding. It is not possible to attribute the main chain conformation of H3 to the particular sequence produced by the join; the structural features of H3 may be due to interactions with HEL and/or with other antibody residues. 相似文献
10.
A V kappa-J kappa junctional change in an antidigoxin recombinant antibody destroys digoxin-binding activity 总被引:3,自引:0,他引:3
N W Hudson R E Bruccoleri L K Steinrauf J A Hamilton M Mudgett-Hunter M N Margolies 《Journal of immunology (Baltimore, Md. : 1950)》1990,145(8):2718-2724
A set of high affinity antidigoxin antibodies were previously identified with high homologous V kappa 1A L chain sequences but were associated with two entirely different VH regions and two dramatically different specificities for digoxin analogs. Antibodies 40-20, 40-60, 40-90, and 40-100 displayed similar binding specificities but differed from that of antibody 26-10. In a previous study using somatic cell fusion for Ig chain recombination we demonstrated that a recombinant antibody consisting of the H chain of antibody 26-10 and the L chain of antibody 40-20 retained digoxin binding and the 26-10 Id, but displayed a binding specificity pattern dominated by the 26-10 H chain donor. In the present study we produced three additional chain recombinant antibodies that contain the 26-10 H chain recombined with each of the L chains of antibodies 40-60, 40-90, and 40-100. All four recombinants expressed the 26-10 Id indistinguishably from the 26-10 antibody. Two of the recombinants (using the 40-60 and 40-90 L chains) bind digoxin; however, the recombinant using the 40-100 L chain failed to bind digoxin. Complete sequence analyses of the 40-20, 40-60, 40-90, and 40-100 VH and VL regions were performed. Antibodies 40-90 and 40-100 have identical VH region sequences but differed only in their L chains at position 96 (proline/leucine). This single difference at the VK-JK junction abolished digoxin binding in the context of one H chain (26-10), but does not cause a significant change in binding in association with the "normal" parental chains 40-90 and 40-100. Thus, structurally closely related VL regions can recombine with different VH regions to form digoxin binding sites of different specificity; in one binding site the identity of a L chain junctional residue is critical whereas in the second binding site that residue is unimportant. Molecular modeling studies revealed major differences between calculated binding site structures for 26-10 when leucine is substituted for proline at position 96 in the 26-10 VL region. 相似文献
11.
V Pascual K Victor D Lelsz M B Spellerberg T J Hamblin K M Thompson I Randen J Natvig J D Capra F K Stevenson 《Journal of immunology (Baltimore, Md. : 1950)》1991,146(12):4385-4391
Cold agglutinins are human autoantibodies, usually of the IgM class, which agglutinate RBC at low temperature. The major subset recognizes the I/i carbohydrate Ag, and many of these antibodies bear cross-reacting idiotypic determinants. An anti-idiotypic mAb that is specific for one of the idiotopes largely confined to cold agglutinins has been used to identify and monitor tumor cells that secrete these molecules in two patients. The tumor cells were immortalized with EBV and the idiotope-positive lines used to investigate the utilization of the VH and VL genes by these antibodies. Nucleotide sequence analysis of the two cold agglutinins (FS-1 and FS-2) revealed the utilization of a single common gene segment, VH4-21. Serologic analysis documented that only human antibodies utilizing the VH4-21 gene segment were reactive in the idiotope assay, other VHIV antibodies as well as a panel of antibodies derived from other VH families being negative. The DH, JH, VK, and JK gene segments of FS-1 and FS-2 were structurally distinct. These data suggest that the structural basis for the cross-reactive idiotope as well as cold agglutinin activity is the VH4-21 gene segment. A nucleotide change in H chain CDR1 of both cold agglutinins results in the substitution of an aspartic acid residue for glycine at position 31, suggesting that this amino acid might be critical to recognition of the red cell Ag. 相似文献
12.
Crystal structure to 2.45 A resolution of a monoclonal Fab specific for the Brucella A cell wall polysaccharide antigen. 总被引:2,自引:1,他引:1
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D. R. Rose M. Przybylska R. J. To C. S. Kayden R. P. Oomen E. Vorberg N. M. Young D. R. Bundle 《Protein science : a publication of the Protein Society》1993,2(7):1106-1113
The atomic structure of an antibody antigen-binding fragment (Fab) at 2.45 A resolution shows that polysaccharide antigen conformation and Fab structure dictated by combinatorial diversity and domain association are responsible for the fine specificity of the Brucella-specific antibody, YsT9.1. It discriminates the Brucella abortus A antigen from the nearly identical Brucella melitensis M antigen by forming a groove-type binding site, lined with tyrosine residues, that accommodates the rodlike A antigen but excludes the kinked structure of the M antigen, as envisioned by a model of the antigen built into the combining site. The variable-heavy (VH) and variable-light (VL) domains are derived from genes closely related to two used in previously solved structures, M603 and R19.9, respectively. These genes combine in YsT9.1 to form an antibody of totally different specificity. Comparison of this X-ray structure with a previously built model of the YsT9.1 combining site based on these homologies highlights the importance of VL:VH association as a determinant of specificity and suggests that small changes at the VL:VH interface, unanticipated in modeling, may cause significant modulation of binding-site properties. 相似文献
13.
The immunoglobulin site(s) that mediates the alternative mechanism of interaction between immunoglobulins and staphylococcal protein A (SpA) was studied by using a monoclonal human IgM. Several IgM fragments were tested for their inhibitory effect in a competitive binding assay of 125I-IgM to SpA. Only those fragments containing Fab mu pieces showed some inhibitory activity. The reactivity of the Fab mu region was retained in some of its subfragments, such as Fv or the VH domain, unlike isolated light chains or VL domains. Furthermore, antibodies specific for the VH domain completely inhibited the SpA-IgM interaction. These results indicate that the alternative SpA-binding site of IgM is located in VH regions. 相似文献
14.
Epidermal growth factor (EGF) domains are found in many proteins, particularly those of the coagulation/fibrinolytic system. We and others have demonstrated that tissue plasminogen activator (t-PA) and prourokinase are modified by the attachment of fucose to equivalent threonine residues within their EGF domains. Factor XII and protein C each contain two EGF domains; in both proteins, the EGF domain nearest the N terminus has a threonine residue in a position homologous to that which is fucosylated in t-PA. In protein C, this site is 3 residues from the position of another post-translational modification, beta-hydroxylation of Asp-71. We isolated peptides containing these sites to determine, primarily by mass spectrometric analysis, the presence of O-linked fucose and/or beta-hydroxyaspartate. We found that factor XII is fully fucosylated at Thr-90. Protein C is unmodified at the equivalent site (Thr-68) and is completely beta-hydroxylated at Asp-71. It has been recently reported that the first EGF domain of human factor VII has O-linked fucose at the equivalent position (Ser-60) (Bjoern, S., Foster, D. C., Thim, L., Wiberg, F. C., Christensen, M., Komiyama, Y., Pedersen, A. H., and Kisiel, W. (1991) J. Biol. Chem. 266, 11051-11057), while it is unmodified at Asp-63 despite having the consensus sequence for beta-hydroxylation at the latter site. These observations raise the possibility that O-linked fucosylation and beta-hydroxylation of EGF domains are mutually exclusive post-translational modifications. 相似文献
15.
K W Harlow D N Smith J A Katzenellenbogen G L Greene B S Katzenellenbogen 《The Journal of biological chemistry》1989,264(29):17476-17485
Radiosequence analysis of peptide fragments of the estrogen receptor (ER) from MCF-7 human breast cancer cells has been used to identify cysteine 530 as the site of covalent attachment of an estrogenic affinity label, ketononestrol aziridine (KNA), and an antiestrogenic affinity label, tamoxifen aziridine (TAZ). ER from MCF-7 cells was covalently labeled with [3H]TAZ or [3H]KNA and purified to greater than 95% homogeneity by immunoadsorbent chromatography. Limit digest peptide fragments, generated by prolonged exposure of the labeled receptor to trypsin, cyanogen bromide, or Staphylococcus aureus V8 protease, were purified to homogeneity by high performance liquid chromatography (HPLC), and the position of the labeled residue was determined by sequential Edman degradation. With both aziridines, the labeled residue was at position 1 in the tryptic peptide, position 2 in the cyanogen bromide peptide, and position 7 in the V8 protease peptide. This localizes the site of labeling to a single cysteine at position 530 in the receptor sequence. The identity of cysteine as the site of labeling was confirmed by HPLC comparison of the TAZ-labeled amino acid (as the phenylthiohydantoin and phenylthiocarbamyl derivatives) and the KNA-labeled amino acid (as the phenylthiocarbamyl derivative) with authentic standards prepared by total synthesis. Cysteine 530 is located in the hormone binding domain of the receptor, near its carboxyl terminus. This location is consistent with earlier studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to analyze the size of the proteolytic fragments containing the covalent labeling sites for TAZ and KNA and the antigen recognition sites for monoclonal antibodies. The fact that both the estrogenic and antiestrogenic affinity labeling agents react covalently with the same cysteine indicates that differences in receptor-agonist and receptor-antagonist complexes do not result in differential covalent labeling of amino acid residues in the hormone binding domain. 相似文献
16.
The regulatory and substrate binding domains of D-3-phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) from Escherichia coli are connected by a single polypeptide strand that contains a Gly-Gly sequence approximately midway between the domains. The potential flexibility of this sequence and its strategic location between major domain structures suggests that it may function in the conformational change leading from effector binding to inhibition of the active site. Site-directed mutagenesis of this region (Gly-336-Gly-337) supports this hypothesis. When bulky side chains were substituted for the glycines at these positions, substantial changes in the ability of serine to inhibit the enzyme were seen with little effect on the activity of the enzyme. The effect of these substitutions could be alleviated by placing a new glycine residue at position 335, immediately flanking the original glycine pair. On the other hand, substituting a glycine at position 338 revealed a critical role for the side chain of Arg-338. This residue may function in stabilizing the conformation about the Gly-Gly turn, resulting in a specific orientation of the adjacent domains relative to each other. Rotation about the phi or psi bonds of either Gly-336 or Gly-337 would have a profound effect on this orientation. The data are consistent with this as a role for the Gly-Gly sequence between the regulatory and substrate binding domains of PGDH. 相似文献
17.
Fatty acid acylation at the single cysteine residue in the cytoplasmic domain of the glycoprotein of vesicular-stomatitis virus. 总被引:4,自引:0,他引:4
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The cysteine residue in the cytoplasmic domain at position 489 of the sequence of the glycoprotein (G protein) isolated from vesicular-stomatitis virions is completely blocked for carboxymethylation. After release of covalently bound fatty acids by hydroxylamine at pH 6.8, this cysteine residue could be specifically labelled by iodo[14C]acetic acid. Reaction products were analysed after specific cleavage of labelled G protein at asparagine-glycine bonds by hydroxylamine at pH 9.3, which generated a C-terminal peptide of Mr 15,300 containing only the single cysteine residue. Bromelain digestion of [3H]palmitic acid-labelled membrane fractions of vesicular-stomatitis-virus-infected baby-hamster kidney cells removed almost completely the 3H radioactivity from the cytoplasmic domain of the G protein, whereas the ectodomain was completely protected by the microsomal membrane. This result indicates that the acylation site of the G protein is exposed on the cytoplasmic side of intracellular membranes. Taken together, both biochemical techniques strongly suggest that the single cysteine-489 residue, which is located six amino acid residues distal to the putative transmembrane domain, is the acylation site. The thioester bond between palmitic acid and the G protein is quite resistant to hydroxylamine treatment (0.32 M at pH 6.8 for 1 h at 37 degrees C) compared with the reactivity of the thioester linkage in palmitoyl-CoA, which is cleaved at relatively low concentrations of hydroxylamine (0.05 M). 相似文献
18.
Jayant B. Udgaonkar Ranabir Das Shachi Gosavi 《Protein science : a publication of the Protein Society》2017,26(10):1994-2002
Rational engineering of a protein to enable domain swapping requires an understanding of the sequence, structural and energetic factors that favor the domain‐swapped oligomer over the monomer. While it is known that the deletion of loops between β‐strands can promote domain swapping, the spliced sequence at the position of the loop deletion is thought to have a minimal role to play in such domain swapping. Here, two loop‐deletion mutants of the non‐domain‐swapping protein monellin, frame‐shifted by a single residue, were designed. Although the spliced sequence in the two mutants differed by only one residue at the site of the deletion, only one of them (YEIKG) promoted domain swapping. The mutant containing the spliced sequence YENKG was entirely monomeric. This new understanding that the domain swapping propensity after loop deletion may depend critically on the chemical composition of the shortened loop will facilitate the rational design of domain swapping. 相似文献
19.
HMG-D is an architecture-specific protein that preferentially binds to DNA containing the dinucleotide TG. 总被引:6,自引:3,他引:3
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M E Churchill D N Jones T Glaser H Hefner M A Searles A A Travers 《The EMBO journal》1995,14(6):1264-1275
The high mobility group (HMG) protein HMG-D from Drosophila melanogaster is a highly abundant chromosomal protein that is closely related to the vertebrate HMG domain proteins HMG1 and HMG2. In general, chromosomal HMG domain proteins lack sequence specificity. However, using both NMR spectroscopy and standard biochemical techniques we show that binding of HMG-D to a single DNA site is sequence selective. The preferred duplex DNA binding site comprises at least 5 bp and contains the deformable dinucleotide TG embedded in A/T-rich sequences. The TG motif constitutes a common core element in the binding sites of the well-characterized sequence-specific HMG domain proteins. We show that a conserved aromatic residue in helix 1 of the HMG domain may be involved in recognition of this core sequence. In common with other HMG domain proteins HMG-D binds preferentially to DNA sites that are stably bent and underwound, therefore HMG-D can be considered an architecture-specific protein. Finally, we show that HMG-D bends DNA and may confer a superhelical DNA conformation at a natural DNA binding site in the Drosophila fushi tarazu scaffold-associated region. 相似文献
20.
Immunoglobulin VH clan and family identity predicts variable domain structure and may influence antigen binding.
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Mammalian immunoglobulin VH families can be grouped into three distinct clans based upon sequence conservation in two of the three framework (FR) intervals. Through replacement/silent site substitution analysis, molecular modeling and mathematical evaluation of known immunoglobulin crystal structures, we demonstrate that this conservation reflects preservation of protein sequence and structure. Each clan contains a characteristic FR 1 interval that is solvent-exposed and structurally separated from the antigen binding site. Families within a clan contain their own unique FR 3 interval that is capable of either influencing the conformation of the antigen binding site or interacting directly with antigen. Our results provide a structural context for theories that address differential use of VH families in the immune response. 相似文献