Structural features of the human salivary mucin, MUC7

Human salivary mucin (MUC7) is characterized by a single polypeptide chain of 357 aa. Detailed analysis of the derived MUC7 peptide sequence reveals five distinct regions or domains: (1) an N-terminal basic, histatin-like domain which has a leucine-zipper segment, (2) a moderately glycosylated domain, (3) six heavily glycosylated tandem repeats each consisting of 23 aa, (4) another heavily glycosylated MUC1- and MUC2-like domain, and (5) a C-terminal leucine-zipper segment. Chemical analysis and semi-empirical prediction algorithms for O-glycosylation suggested that 86/105 (83%) Ser/Thr residues were O-glycosylated with the majority located in the tandem repeats. The high (～25%) proline content of MUC7 including 19 diproline segments suggested the presence of polyproline type structures. CD studies of natural and synthetic diproline-rich peptides and glycopeptides indicated that polyproline type structures do play a significant role in the conformational dynamics of MUC7. In addition, crystal structure analysis of a synthetic diproline segment (Boc-Ala-Pro-OBzl) revealed a polyproline type II extended structure. Collectively, the data indicate that the polyproline type II structure, dispersed throughout the tandem repeats, may impart a stiffening of the backbone and could act in consort with the glycosylated segments to keep MUC7 in a semi-rigid, rod shaped conformation resembling a ‘bottle-brush’ model.     

Interactions between the leucine-zipper motif of cGMP-dependent protein kinase and the C-terminal region of the targeting subunit of myosin light chain phosphatase

Nitric oxide induces vasodilation by elevating the production of cGMP, an activator of cGMP-dependent protein kinase (PKG). PKG subsequently causes smooth muscle relaxation in part via activation of myosin light chain phosphatase (MLCP). To date, the interaction between PKG and the targeting subunit of MLCP (MYPT1) is not fully understood. Earlier studies by one group of workers showed that the binding of PKG to MYPT1 is mediated by the leucine-zipper motifs at the N and C termini, respectively, of the two proteins. Another group, however, reported that binding of PKG to MYPT1 did not require the leucine-zipper motif of MYPT1. In this work we fully characterized the interaction between PKG and MYPT1 using biophysical techniques. For this purpose we constructed a recombinant PKG peptide corresponding to a predicted coiled coil region that contains the leucine-zipper motif. We further constructed various C-terminal MYPT1 peptides bearing various combinations of a predicted coiled coil region, extensions preceding this coiled coil region, and the leucine-zipper motif. Our results show, firstly, that while the leucine-zipper motif at the N terminus of PKG forms a homodimeric coiled coil, the one at the C terminus of MYPT1 is monomeric and non-helical. Secondly, the leucine-zipper motif of PKG binds to that of MYPT1 to form a heterodimer. Thirdly, when the leucine-zipper motif of MYPT1 is absent, the PKG leucine-zipper motif binds to the coiled coil region and upstream segments of MYPT1 via formation of a heterotetramer. These results provide rationalization of some of the findings by others using alternative binding analyses.     

Localization of the fourth membrane spanning domain as a ligand binding site in the human platelet alpha 2-adrenergic receptor

The human platelet alpha 2-adrenergic receptor is an integral membrane protein which binds epinephrine. The gene for this receptor has been cloned, and the primary structure is thus known [Kobilka et al. (1987) Science 238, 650-656]. A model of its secondary structure predicts that the receptor has seven transmembrane spanning domains. By covalent labeling and peptide mapping, we have identified a region of the receptor that is directly involved with ligand binding. Partially purified preparations of the receptor were covalently radiolabeled with either of two specific photoaffinity ligands: [3H]SKF 102229 (an antagonist) or p-azido[3H]clonidine (an agonist). The radiolabeled receptors were then digested with specific endopeptidases, and peptides containing the covalently bound radioligands were identified. Lysylendopeptidase treatment of [3H]SKF 102229 labeled receptor yielded one peptide of Mr 2400 as the product of a complete digest. Endopeptidase Arg-C gave a labeled peptide of Mr 4000, which was further digested to the Mr 2400 peptide by additional treatment with lysylendopeptidase. Using p-azido[3H]clonidine-labeled receptor, a similar Mr 2400 peptide was obtained by lysylendopeptidase cleavage. This Mr 2400 peptide corresponds to the fourth transmembrane spanning domain of the receptor. These data suggest that this region forms part of the ligand binding domain of the human platelet alpha 2-adrenergic receptor.     

Identification and characterization of PKNbeta, a novel isoform of protein kinase PKN: expression and arachidonic acid dependency are different from those of PKNalpha.

The cDNA clone encoding a novel isoform of protein kinase PKN, termed PKNbeta, was isolated from a HeLa cDNA library. PKNbeta had high sequence homology with PKNalpha, originally isolated PKN, especially in the repeats of charged amino acid-rich region with leucine-zipper like sequences (CZ region/HR1), in the carboxyl-terminal catalytic domain, and in approximately 130 amino acid stretch (D region/HR2), located between CZ region/HR1 and the catalytic domain. However, the amino acid sequence of PKNbeta differed from that of PKNalpha in the region immediately amino-terminal to the catalytic domain, which contained two distinct proline-rich sequences consistent with the class II consensus sequence, PXXPXR, for binding to SH3 domain. Distribution of PKNbeta differed from that of PKNalpha in the following two respects: (1) Northern blotting indicated that PKNbeta mRNA could not be detected in human adult tissues, but was expressed abundantly in human cancer cell lines; (2) immunochemical analysis indicated that PKNbeta localized in nucleus and perinuclear Golgi apparatus, and was almost absent in cytoplasmic region in NIH3T3 cells. Recombinant PKNbeta expressed in COS7 cells displayed autophosphorylation and peptide kinase activity, but was found to be significantly less responsive to arachidonic acid than PKNalpha. The identification of this novel isoform underscores the diversity of PKN signaling pathway.     

Structure of the anchor-domain of myristoylated and non-myristoylated HIV-1 Nef protein.

Negative factor (Nef) is a regulatory myristoylated protein of human immunodeficiency virus (HIV) that has a two-domain structure consisting of an anchor domain and a core domain separated by a specific cleavage site of the HIV proteases. For structural analysis, the HIV-1 Nef anchor domain (residues 2-57) was synthesized with a myristoylated and non-myristoylated N terminus. The structures of the two peptides were studied by1H NMR spectroscopy and a structural model was obtained by restrained molecular dynamic simulations. The non-myristoylated peptide does not have a unique, compactly folded structure but occurs in a relatively extended conformation. The only rather well-defined canonical secondary structure element is a short two-turn alpha-helix (H2) between Arg35 and Gly41. A tendency for another helical secondary structure element (H1) can be observed for the arginine-rich region (Arg17 to Arg22). Myristoylation of the N-terminal glycine residue leads to stabilization of both helices, H1 and H2. The first helix in the arginine-rich region is stabilized by the myristoylation and now contains residues Pro14 to Arg22. The second helix appears to be better defined and to contain more residues (Ala33 to Gly41) than in the absence of myristoylation. In addition, the hydrophobic N-terminal myristic acid residue interacts closely with the side-chain of Trp5 and thereby forms a loop with Gly2, Gly3 and Lys4 in the kink region. This interaction could possibly be disturbed by phosphorylation of a nearby serine residue, and modifiy the characteristic membrane interactions of the HIV-1 Nef anchor domain.     

非核糖体肽合成酶基因腺苷酰化结构域序列克隆及分析

微生物许多非核糖体肽类次生代谢产物主要是由非核糖体肽合成酶(NRPS)催化合成。参考Gontang发布的非核糖体肽合成酶(NRPS)通用引物设计扩增NRPS腺苷酰化结构域基因序列的特异引物,从海洋链霉菌L1的基因组DNA中扩增获得一个715 bp的NRPS基因序列。测序结果及比对分析表明该片段属于NRPS腺苷酰化结构域部分序列。对其拟翻译的氨基酸序列组成成分、理化性质进行分析,显示其包含AFD class I超基因家族核心结合区,为NRPS腺苷酰化结构域(A结构域)所在区域。对氨基酸序列的二级结构预测和三级结构模拟,发现与数据库中肠菌素合酶F组分的结构相似。为后续研究A结构域的特异性及完整NRPS基因簇克隆提供了参考。     

Solution structure of the sixth transmembrane helix of the G-protein-coupled receptor, rhodopsin

Low resolution electron density maps have revealed the general orientation of the transmembrane helices of rhodopsin. However, high resolution structural information for the transmembrane domain of the G-protein-coupled receptor, rhodopsin, is as yet unavailable. In this study, a high resolution solution structure is reported for a 15 residue portion of the sixth transmembrane helix of rhodopsin (rhovih) as a free peptide. Helix 6 is one of the transmembrane helices of rhodopsin that contains a proline (amino acid residue 267) and the influence of this proline on the structure of this transmembrane domain was unknown. The structure obtained shows an alpha-helix through most of the sequence. The proline apparently induces only a modest distortion in the helix. Previously, the structure of the intradiskal loop connected to helix 6 was solved. The sequence of this loop contained five residues in common (residues 268-272) with the peptide reported here from the rhovih. The five residues in common between these two structures were superimposed to connect these two structures. The superposition showed a root mean square deviation of 0.2 A. Thus, this five residue sequence formed the same structure in both peptides, indicating that the structure of this region is governed primarily by short range interactions.     

Conformational analysis by CD and NMR spectroscopy of a peptide encompassing the amphipathic domain of YopD from Yersinia.

To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III secretion machine that permits the translocation of several anti-host factors into the cytosol of target eukaryotic cells. Secreted YopD is essential for this process. Pre-secretory stabilization of YopD is mediated by an interaction with its cognate chaperone, LcrH. YopD possesses LcrH binding domains located in the N-terminus and in a predicted amphipathic domain located near the C-terminus. This latter domain is also critical for Yersinia virulence. In this study, we designed synthetic peptides encompassing the C-terminal amphipathic domain of YopD. A solution structure of YopD278-300, a peptide that strongly interacted with LcrH, was obtained by NMR methods. The structure is composed of a well-defined amphipathic alpha helix ranging from Phe280 to Tyr291, followed by a type I beta turn between residues Val292 and His295. The C-terminal truncated peptides, YopD278-292 and YopD271-292, lacked helical structure, implicating the beta turn in helix stability. An interaction between YopD278-300 and its cognate chaperone, LcrH, was observed by NMR through line-broadening effects and chemical shift differences between the free peptide and the peptide-LcrH complex. These effects were not observed for the unstructured peptide, YopD278-292, which confirms that the alpha helical structure of the YopD amphipathic domain is a critical binding region of LcrH.     

Protein tyrosine phosphatase SHP-1 specifically recognizes C-terminal residues of its substrates via helix alpha0

The catalytic domain of protein tyrosine phosphatase SHP-1 possesses distinct substrate specificity. It recognizes the P-3 to P-5 residues of its substrates via the beta5-loop-beta6 region. To study the substrate specificity further, we determined the structure of the catalytic domain of SHP-1 (C455S) complexed with a less-favorable-substrate peptide originated from SIRPalpha. The complex has disordered N-terminal peptide structure and reduced interactions between the N-terminal peptide and the beta5-loop-beta6 region. This could be the basis for the lower affinity of peptide pY(427) for the catalytic domain of SHP-1. In addition, by comparing the SHP-1/less-favorable peptide complex structure with the SHP-1/substrate complex structures, we identified a novel substrate-recognition site in the catalytic domain of SHP-1. This site was formed by helix alpha0 and the alpha5-loop-alpha6 motif of SHP-1, and specifically bound residues at the P + 4 and further C-terminal positions of peptide substrates.     

Conformation and interaction with the membrane models of the amino-terminal peptide of influenza virus hemagglutinin HA2 at fusion pH.

Conformations of a 48-mer peptide corresponding to the amino-terminal region of influenza HA2 in aqueous and membranous environments were studied. In aqueous solution the peptide was found to be oligomeric and its helicity was enhanced at higher concentrations. The conformation in phospholipid bilayer and insertion depth into the sodium dodecyl sulfate (SDS) micelle for the fusion peptide were in line with those determined for the amino-terminal 25-mer analog. The turn of residues 28-31 found in the crystal structure of hemagglutinin at neutral pH persisted in the presence of SDS at pH 5.0. Except for the turn, conformational lability of the amino portion of HA2 is suggested by comparison of the secondary structure determined herein with that obtained with the influenza fusion protein crystallized in the aqueous phase at neutral pH. The backbone amide proton exchange experiment suggested an interaction with the micellar surface for the segment carboxy-terminal to the fusion peptide domain.     

Solution structure of the N-terminal amphitropic domain of Escherichia coli glucose-specific enzyme IIA in membrane-mimetic micelles

The N-terminal domain of enzyme IIA(Glc) of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system confers amphitropism to the protein, allowing IIA(Glc) to shuttle between the cytoplasm and the membrane. To further understand this amphitropic protein, we have elucidated, by NMR spectroscopy, the solution structure of a synthetic peptide corresponding to the N-terminal domain of IIA(Glc). In water, this peptide is predominantly disordered, consistent with previous data obtained in the absence of membranes. In detergent micelles of dihexanoylphosphatidylglycerol (DHPG) or sodium dodecylsulfate (SDS), however, residues Phe 3-Val 10 of the peptide adopt a helical conformation in the ensemble of structures calculated on the basis of NOE-derived distance restraints. The root mean square deviations for superimposing the backbone atoms of the helical region are 0.18 A in DHPG and 0.22 A in SDS. The structure, chemical shifts, and spin-spin coupling constants all indicate that, of the four lysines in the N-terminal domain of IIA(Glc), only Lys 5 and Lys 7 in the amphipathic helical region interact with DHPG. In addition, the peptide-detergent interactions were investigated using intermolecular NOESY experiments. The aliphatic chains of anionic detergents DHPG, SDS, and 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt (DSS) all showed intermolecular NOE cross-peaks to the peptide, providing direct evidence for the putative membrane anchor of IIA(Glc) in binding to the membrane-mimicking micelles.     

Molecular modeling of the amyloid-beta-peptide using the homology to a fragment of triosephosphate isomerase that forms amyloid in vitro

The main component of the amyloid senile plaques found in Alzheimer's brain is the amyloid-beta-peptide (A beta), a proteolytic product of a membrane precursor protein. Previous structural studies have found different conformations for the A beta peptide depending on the solvent and pH used. In general, they have suggested an alpha-helix conformation at the N-terminal domain and a beta-sheet conformation for the C-terminal domain. The structure of the complete A beta peptide (residues 1-40) solved by NMR has revealed that only helical structure is present in A beta. However, this result cannot explain the large beta-sheet A beta aggregates known to form amyloid under physiological conditions. Therefore, we investigated the structure of A beta by molecular modeling based on extensive homology using the Smith and Waterman algorithm implemented in the MPsrch program (Blitz server). The results showed a mean value of 23% identity with selected sequences. Since these values do not allow a clear homology to be established with a reference structure in order to perform molecular modeling studies, we searched for detailed homology. A 28% identity with an alpha/beta segment of a triosephosphate isomerase (TIM) from Culex tarralis with an unsolved three-dimensional structure was obtained. Then, multiple sequence alignment was performed considering A beta, TIM from C.tarralis and another five TIM sequences with known three-dimensional structures. We found a TIM segment with secondary structure elements in agreement with previous experimental data for A beta. Moreover, when a synthetic peptide from this TIM segment was studied in vitro, it was able to aggregate and to form amyloid fibrils, as established by Congo red binding and electron microscopy. The A beta model obtained was optimized by molecular dynamics considering ionizable side chains in order to simulate A beta in a neutral pH environment. We report here the structural implications of this study.     

Structural insight into the interaction between the p55 PDZ domain and glycophorin C

p55, a member of the membrane-associated guanylate kinase family, includes a PDZ domain that specifically interacts with the C-terminal region of glycophorin C in the ternary complex of p55, protein 4.1 and glycophorin C. Here we present the first NMR-derived complex structure of the p55 PDZ domain and the C-terminal peptide of glycophorin C, obtained by using a threonine to cysteine (T85C) mutant of the p55 PDZ domain and a phenylalanine to cysteine (F127C) mutant of the glycophorin C peptide. Our NMR results revealed that the two designed mutant molecules retain the specific interaction manner that exists between the wild type molecules and can facilitate the structure determination by NMR, due to the stable complex formation via an intermolecular disulfide bond. The complex structure provides insight into the specific interaction of the p55 PDZ domain with the two key residues, Ile128 and Tyr126, of glycophorin C.     

Structure of the C-terminal phosphotyrosine interaction domain of Fe65L1 complexed with the cytoplasmic tail of amyloid precursor protein reveals a novel peptide binding mode

Fe65L1, a member of the Fe65 family, is an adaptor protein that interacts with the cytoplasmic domain of Alzheimer amyloid precursor protein (APP) through its C-terminal phosphotyrosine interaction/phosphotyrosine binding (PID/PTB) domain. In the present study, the solution structures of the C-terminal PID domain of mouse Fe65L1, alone and in complex with a 32-mer peptide (DAAVTPEERHLSKMQQNGYENPTYKFFEQMQN) derived from the cytoplasmic domain of APP, were determined using NMR spectroscopy. The C-terminal PID domain of Fe65L1 alone exhibits a canonical PID/PTB fold, whereas the complex structure reveals a novel mode of peptide binding. In the complex structure, the NPTY motif forms a type-I beta-turn, and the residues immediately N-terminal to the NPTY motif form an antiparallel beta-sheet with the beta5 strand of the PID domain, the binding mode typically observed in the PID/PTB.peptide complex. On the other hand, the N-terminal region of the peptide forms a 2.5-turn alpha-helix and interacts extensively with the C-terminal alpha-helix and the peripheral regions of the PID domain, representing a novel mode of peptide binding that has not been reported previously for the PID/PTB.peptide complex. The indispensability of the N-terminal region of the peptide for the high affinity of the PID-peptide interaction is consistent with NMR titration and isothermal calorimetry data. The extensive binding features of the PID domain of Fe65L1 with the cytoplasmic domain of APP provide a framework for further understanding of the function, trafficking, and processing of APP modulated by adapter proteins.     

A peptide analog of the calmodulin-binding domain of myosin light chain kinase adopts an alpha-helical structure in aqueous trifluoroethanol.

A 22-residue synthetic peptide encompassing the calmodulin (CaM)-binding domain of skeletal muscle myosin light chain kinase was studied by two-dimensional NMR and CD spectroscopy. In water the peptide does not form any regular structure; however, addition of the helix-inducing solvent trifluoroethanol (TFE) causes it to form an alpha-helical structure. The proton NMR spectra of this peptide in 25% and 40% TFE were assigned by double quantum-filtered J-correlated spectroscopy, total correlation spectroscopy, and nuclear Overhauser effect correlated spectroscopy spectra. In addition, the alpha-carbon chemical shifts were obtained from (1H,13C)-heteronuclear multiple quantum coherence spectra. The presence of numerous dNN(i, i + 1), d alpha N(i, i + 3), and d alpha beta(i, i + 3) NOE crosspeaks indicates that an alpha-helix can be formed from residues 3 to 20; this is further supported by the CD data. Upfield alpha-proton and downfield alpha-carbon shifts in this region of the peptide provide further support for the formation of an alpha-helix. The helix induced by TFE appears to be similar to that formed upon binding of the peptide to CaM.     

Sequence analysis of phospholamban. Identification of phosphorylation sites and two major structural domains

Phospholamban is a regulatory protein in cardiac sarcoplasmic reticulum that is phosphorylated by cAMP- and Ca2+/calmodulin-dependent protein kinase activities. In this report, we present the partial amino acid sequence of canine cardiac phospholamban and the identification of the sites phosphorylated by these two protein kinases. Gas-phase protein sequencing was used to identify 20 NH2-terminal residues. Overlap peptides produced by trypsin or papain digestion extended the sequence 16 residues to give the following primary structure: Ser-Ala-Ile-Arg-Arg-Ala-Ser-Thr-Ile-Glu-Met-Pro-Gln-Gln-Ala- Arg-Gln-Asn-Leu-Gln-Asn-Leu-Phe-Ile-Asn-Phe-(Cys)-Leu-Ile-Leu-Ile-(Cys)- Leu-Leu-Leu-Ile-. Phospholamban phosphorylated by either cAMP-dependent or Ca2+/calmodulin-dependent protein kinase was cleaved with trypsin, and the major phosphorylated peptide (comprising greater than 70% of the incorporated 32P label) was purified by reverse-phase high performance liquid chromatography. The identical sequence was revealed for the radioactive peptide obtained from phospholamban phosphorylated by either kinase: Arg-Ala-Ser-Thr-Ile-Glu-Met-Pro-Gln-Gln-. The adjacent residues Ser7 and Thr8 of phospholamban were identified as the unique sites phosphorylated by cAMP- and Ca2+/calmodulin-dependent protein kinases, respectively. These results establish that phospholamban is an oligomer of small, identical polypeptide chains. A hydrophilic, cytoplasmically oriented NH2-terminal domain on each monomer contains the unique, adjacent residues phosphorylated by cAMP- and Ca2+/calmodulin-dependent protein kinase activities. Analysis by hydropathic profiling and secondary structure prediction suggests that phospholamban monomers also contain a hydrophobic domain, which could form amphipathic helices sufficiently long to traverse the sarcoplasmic reticulum membrane. A model of phospholamban as a pentamer is presented in which the amphipathic alpha-helix of each monomer is a subunit of the pentameric membrane-anchored domain, which is comprised of an exterior hydrophobic surface and an interior hydrophilic region containing polar side chains.