A role for prolyl 3-hydroxylase 2 in post-translational modification of fibril-forming collagens

The fibrillar collagen types I, II, and V/XI have recently been shown to have partially 3-hydroxylated proline (3Hyp) residues at sites other than the established primary Pro-986 site in the collagen triple helical domain. These sites showed tissue specificity in degree of hydroxylation and a pattern of D-periodic spacing. This suggested a contributory role in fibril supramolecular assembly. The sites in clade A fibrillar α1(II), α2(V), and α1(I) collagen chains share common features with known prolyl 3-hydroxylase 2 (P3H2) substrate sites in α1(IV) chains implying a role for this enzyme. We pursued this possibility using the Swarm rat chondrosarcoma cell line (RCS-LTC) found to express high levels of P3H2 mRNA. Mass spectrometry determined that all the additional candidate 3Hyp substrate sites in the pN type II collagen made by these cells were highly hydroxylated. In RNA interference experiments, P3H2 protein synthesis was suppressed coordinately with prolyl 3-hydroxylation at Pro-944, Pro-707, and the C-terminal GPP repeat of the pNα1(II) chain, but Pro-986 remained fully hydroxylated. Furthermore, when P3H2 expression was turned off, as seen naturally in cultured SAOS-2 osteosarcoma cells, full 3Hyp occupancy at Pro-986 in α1(I) chains was unaffected, whereas 3-hydroxylation of residue Pro-944 in the α2(V) chain was largely lost, and 3-hydroxylation of Pro-707 in α2(V) and α2(I) chains were sharply reduced. The results imply that P3H2 has preferred substrate sequences among the classes of 3Hyp sites in clade A collagen chains.     

Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1.5 gating by multiple phosphorylation sites

The cardiac Na(+) channel Na(V)1.5 current (I(Na)) is critical to cardiac excitability, and altered I(Na) gating has been implicated in genetic and acquired arrhythmias. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is up-regulated in heart failure and has been shown to cause I(Na) gating changes that mimic those induced by a point mutation in humans that is associated with combined long QT and Brugada syndromes. We sought to identify the site(s) on Na(V)1.5 that mediate(s) the CaMKII-induced alterations in I(Na) gating. We analyzed both CaMKII binding and CaMKII-dependent phosphorylation of the intracellularly accessible regions of Na(V)1.5 using a series of GST fusion constructs, immobilized peptide arrays, and soluble peptides. A stable interaction between δ(C)-CaMKII and the intracellular loop between domains 1 and 2 of Na(V)1.5 was observed. This region was also phosphorylated by δ(C)-CaMKII, specifically at the Ser-516 and Thr-594 sites. Wild-type (WT) and phosphomutant hNa(V)1.5 were co-expressed with GFP-δ(C)-CaMKII in HEK293 cells, and I(Na) was recorded. As observed in myocytes, CaMKII shifted WT I(Na) availability to a more negative membrane potential and enhanced accumulation of I(Na) into an intermediate inactivated state, but these effects were abolished by mutating either of these sites to non-phosphorylatable Ala residues. Mutation of these sites to phosphomimetic Glu residues negatively shifted I(Na) availability without the need for CaMKII. CaMKII-dependent phosphorylation of Na(V)1.5 at multiple sites (including Thr-594 and Ser-516) appears to be required to evoke loss-of-function changes in gating that could contribute to acquired Brugada syndrome-like effects in heart failure.     

Engineered zinc-binding sites confirm proximity and orientation of transmembrane helices I and III in the human serotonin transporter

The human serotonin transporter (hSERT) regulates neurotransmission by removing released serotonin (5-HT) from the synapse. Previous studies identified residues in SERT transmembrane helices (TMHs) I and III as interaction sites for substrates and antagonists. Despite an abundance of data supporting a 12-TMH topology, the arrangement of the TMHs in SERT and other biogenic amine transporters remains undetermined. A high-resolution structure of a bacterial leucine transporter that demonstrates homology with SERT has been reported, thus providing the basis for the development of a SERT model. Zn2+-binding sites have been utilized in transporters and receptors to define experimentally TMH proximity. Focusing on residues near the extracellular ends of hSERT TMHs I and III, we engineered potential Zn2+-binding sites between V102 or W103 (TMH I) and I179-L184 (TMH III). Residues were mutated to either histidine or cysteine. TMH I/III double mutants were constructed from functional TMH I mutants, and Zn2+ sensitivity was assessed. Dose-response assays suggest an approximately twofold increase in sensitivity to Zn2+ inhibition at the hSERT V102C/M180C and approximately fourfold at the V102C/I179C mutant compared to the hSERT V102C single mutant. We propose that the increased sensitivity to Zn2+ confirms the proximity and the orientation of TMHs I and III in the membrane. Homology modeling of the proposed Zn2+-binding sites using the coordinates of the Aquifex aeolicus leucine transporter structure provided a structural basis for interpreting the results and developing conclusions.     

SH2 domains from suppressor of cytokine signaling-3 and protein tyrosine phosphatase SHP-2 have similar binding specificities

Suppressor of cytokine signaling-3 (SOCS-3) and the protein tyrosine phosphatase SHP-2 both regulate signaling by cytokines of the interleukin-6 family, and this is dependent upon recruitment to tyrosine 757 in the shared cytokine receptor subunit gp130. To better explore the overlap in ligand binding specificities exhibited by these two signaling regulators, we have mapped the phosphopeptide binding preferences of the SH2 domains from SOCS-3 and SHP-2. Degenerate phosphopeptide libraries were screened against recombinantly produced SH2 domains to determine the sequences of optimal phosphopeptide ligands. We found that the consensus ligand binding motif for SOCS-3 was pY-(S/A/V/Y/F)-hydrophobic-(V/I/L)-hydrophobic-(H/V/I/Y), while the consensus motif for SHP-2 was pY-(S/T/A/V/I)-X-(V/I/L)-X-(W/F). We validated these data through the design of phosphopeptide ligands based on the consensus motifs and found that these bound to SOCS-3 and SHP-2 with high affinity. Finally, we have compared the affinity of SOCS-3 for binding to phosphopeptides representing putative docking sites in the gp130, leptin and erythropoietin receptors. While SOCS-3 binds with much higher affinity to a gp130 phosphopeptide than to phosphopeptides derived from the other receptors, multiple SOCS-3 binding sites are predicted to exist in the leptin and erythropoietin receptors which may compensate for weaker binding to individual sites.     

Crystal structure of human annexin I at 2.5 A resolution.

cDNA coding for N-terminally truncated human annexin I, a member of the family of Ca(2+)-dependent phospholipid binding proteins, has been cloned and expressed in Escherichia coli. The expressed protein is biologically active, and has been purified and crystallized in space group P2(1)2(1)2(1) with cell dimensions a = 139.36 A, b = 67.50 A, and c = 42.11 A. The crystal structure has been determined by molecular replacement at 3.0 A resolution using the annexin V core structure as the search model. The average backbone deviation between these two structures is 2.34 A. The structure has been refined to an R-factor of 17.7% at 2.5 A resolution. Six calcium sites have been identified in the annexin I structure. Each is located in the loop region of the helix-loop-helix motif. Two of the six calcium sites in annexin I are not occupied in the annexin V structure. The superpositions of the corresponding loop regions in the four domains show that the calcium binding loops in annexin I can be divided into two classes: type II and type III. Both classes are different from the well-known EF-hand motif (type I).     

Probes for imidazoline binding sites: synthesis and evaluation of a selective, irreversible I2 ligand

An irreversible ligand (7) has been prepared based on the selective I2 ligand 2-BFI. Compound 7 displayed high affinity and selectivity for I2-sites and has been shown to irreversibly bind to these sites in rat brain. Compound 7 should, therefore, prove an invaluable tool for the further elucidation of I2-site function.     

Identification and characterization of the imidazoline I2b-binding sites in the hamster brown adipose tissue as a study model for imidazoline receptors

The imidazoline-type compound, MPV-1743, has been found to activate nonshivering thermogenesis (NST) in brown adipose tissue (BAT) of the genetically obese Zucker rats. The regulation of NST in BAT is linked to the catecholamine metabolism, and the imidazoline I2-binding sites have been found on the monoamine oxidase, a catecholamine metabolising enzyme. In this study, the I2-binding sites of hamster BAT have been characterised using a receptor binding assay with 3H-idazoxan as a radioligand, and the interaction of MPV-1743 with these I2-binding sites has been studied using the enantiomers of MPV 1743, that is, MPV 2088 and MPV 2089. Cirazoline was used to determine the specific binding of 3H-idazoxan to the imidazoline I2-binding sites. Rauwolscine was added in the 3H-idazoxan binding assay in order to inhibit any binding to potential alpha2-adrenergic sites. In the presence of rauwolscine mask 3H-Idazoxan labelled a population of non-adrenergic binding sites expressing the properties of the imidazoline I2b-receptor subtype similar to that found in the rat liver (cirazoline > guanabenz = amiloride > clonidine). The binding of 3H-idazoxan to the I2b-binding sites could be displaced by the imidazole compounds with the following affinities: detomidine (KiHigh 9.2 nM; KiLow 3200 nM), MPV-2088 (KiHigh 19 nM; IKiLow 760 nM) and MPV-2089 (KiHigh 190 nM; KiLow 1300 nM), atipamezole (3500 nM) and dexmedetomidine (Ki 8400 nM). These results have shown that the hamster BAT contains the imidazoline I2b-binding sites with heterogeneous binding properties for some test compounds. In addition, the enantiomers of MPV 1743, that is, MPV 2088 and MPV 2089, had high affinity to these BAT imidazoline I2b-binding sites. Therefore, it is suggested that the regulation of NST in the hamster BAT may be an attractive model to study the role of imidazoline I2b-binding sites.     

Amino-acid sequence of ribonuclease T2 from Aspergillus oryzae

The amino acid sequence of ribonuclease T2 (RNase T2) from Aspergillus oryzae has been determined. This has been achieved by analyzing peptides obtained by digestions with Achromobacter lyticus protease I, Staphylococcus aureus V8 protease, and alpha-chymotrypsin of two large cyanogen bromide peptides derived from the reduced and S-carboxymethylated or S-aminoethylated protein. Digestion with A. lyticus protease I was successfully used to degrade the N-terminal half of the S-aminoethylated protein at cysteine residues. RNase T2 is a glycoprotein consisting of 239 amino acid residues with a relative molecular mass of 29,155. The sugar content is 7.9% (by mass). Three glycosylation sites were determined at Asns 15, 76 and 239. Apparently RNase T2 has a very low degree of sequence similarity with RNase T1, but a considerable similarity is observed around the amino acid residues involved in substrate recognition and binding in RNase T1. These similar residues may be important for the catalytic activity of RNase T2.     

Prevalence of p.V37I Variant of GJB2 in Mild or Moderate Hearing Loss in a Pediatric Population and the Interpretation of Its Pathogenicity

A p.V37I variant of GJB2 has been reported from subjects with moderate or slight hearing loss especially in East Asian populations. This study aimed to estimate the prevalence of the p.V37I variant among such subjects and prove, epidemiologically, its pathogenic potential to cause mild hearing loss. A total of 380 subjects from 201 families with hearing loss were enrolled. From them, 103 families were selected who had autosomal recessive inheritance or sporadic occurrence of hearing loss and who were younger than 15 years old. GJB2 sequencing was carried out for the probands of all 103 families. The prevalence of the p.V37I variant was compared between the subtle, mild or moderate hearing loss (group I) and the severe or profound hearing loss (group II) groups. Where possible, a targeted next generation sequencing of 82 deafness genes was performed from the p.V37I carrier to exclude the existence of other pathogenic genes. Five (4.8%) of 103 probands were found to carry p.V37I. The carrier frequency of p.V37I among group I (18.2%) was significantly higher than that of group II (1.2%) or the reported Korean normal hearing control group (1.0%). Detection of the p.V37I variant of GJB2 in 18.2% of Koreans with mild hearing loss strongly suggests its contribution to the pathogenesis of milder hearing loss, which might justify sequencing of GJB2 from these subjects in the Korean population.     

Mapping of genomic DNA loop organization in a 500-kilobase region of the Drosophila X chromosome by the topoisomerase II-mediated DNA loop excision protocol.

The recently developed procedure of chromosomal DNA loop excision by topoisomerase II-mediated DNA cleavage at matrix attachment sites (S. V. Razin, R. Hancock, O. Iarovaia, O. Westergaard, I. Gromova, and G. P. Georgiev, Cold Spring Harbor Symp. Quant. Biol. 58:25-35, 1993; I. I. Gromova, B. Thompsen, and S. V. Razin, Proc. Natl. Acad. Sci. USA 92:102-106, 1995) has been employed for mapping the DNA loop anchorage sites in a 500-kb region of the Drosophila melanogaster X chromosome. Eleven anchorage sites delimiting 10 DNA loops ranging in size from 20 to 90 kb were found within this region. Ten of these 11 anchorage sites colocalize with previously mapped scaffold attachment regions. However, a number of other scaffold attachment regions are found to be located in loop DNA.     

High‐resolution structural analysis shows how different crystallographic environments can induce alternative modes of binding of a phosphotyrosine peptide to the SH2 domain of Fer tyrosine kinase

Fes and Fes‐related (Fer) protein tyrosine kinases (PTKs) comprise a subfamily of nonreceptor tyrosine kinases characterized by a unique multidomain structure composed of an N‐terminal Fer/CIP4 homology‐Bin/Amphiphysin/Rvs (F‐BAR) domain, a central Src homology 2 (SH2) domain, and a C‐terminal PTK domain. Fer is ubiquitously expressed, and upregulation of Fer has been implicated in various human cancers. The PTK activity of Fes has been shown to be positively regulated by the binding of phosphotyrosine‐containing ligands to the SH2 domain. Here, the X‐ray crystal structure of human Fer SH2 domain bound to a phosphopeptide that has D‐E‐pY‐E‐N‐V‐D sequence is reported at 1.37 å resolution. The asymmetric unit (ASU) contains six Fer‐phosphopeptide complexes, and the structure reveals three distinct binding modes for the same phosphopeptide. At four out of the six binding sites in the ASU, the phosphopeptide binds to Fer SH2 domain in a type I β‐turn conformation, and this could be the optimal binding mode of this phosphopeptide. At the other two binding sites in the ASU, it appears that spatial proximity of neighboring SH2 domains in the crystal induces alternative modes of binding of this phosphopeptide.     

Construction and characterization of a spectral probe mutant of troponin C: application to analyses of mutants with increased Ca2+ affinity.

A spectral probe mutant (F29W) of chicken skeletal muscle troponin C (TnC) has been prepared in which Phe-29 has been substituted by Trp. Residue 29 is at the COOH-terminal end of the A helix immediately adjacent to the Ca2+ binding loop of site I (residues 30-41) of the regulatory N domain. Since this protein is naturally devoid of Tyr and Trp, spectral features can be assigned unambiguously to the single Trp. The fluorescent quantum yield at 336 nm is increased almost 3-fold in going from the Ca(2+)-free state to the 4Ca2+ state with no change in the wavelength of maximum emission. Comparisons of the Ca2+ titration curves of the change in far-UV CD and fluorescence emission indicated that the latter was associated only with the binding of 2Ca2+ to the regulatory sites I and II. No change in fluorescence was detected by titration with Mg2+. The Ca(2+)-induced transitions of both the N and C domains were highly cooperative. Addition of Ca2+ also produced a red shift in the UV absorbance spectrum and a reduction in positive ellipticity as monitored by near-UV CD measurements. The fluorescent properties of F29W were applied to an investigation of five double mutants: F29W/V45T, F29W/M46Q, F29W/M48A, F29W/L49T, and F29W/M82Q. Ca2+ titration of their fluorescent emissions indicated in each case an increased Ca2+ affinity of their N domains. The magnitude of these changes and the decreased cooperativity observed between Ca2+ binding sites I and II for some of the mutants are discussed in terms of the environment of the mutated residues in the 2Ca2+ and modeled 4Ca2+ states.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Alkylation of 2-Cyclopenten-2-ol-1-one and Cyclotene through Their Ketimine Derivative

A new synthetic method of cyclotene (3-methyl-2-cyclopenten-2-ol-1-one) (I) and its derivatives has been investigated. The reaction of 2-cyclopenten-2-ol-1-one and aniline in toluene gave the corresponding ketimine derivative (V) in good yield. The methylation of (V) afforded (I) and 5,5-dimethyl-2-cyclopenten-2-ol-1-one (II) as the major reaction products, and 3,5-dimethyl-2-cyclopenten-2-ol-1-one (III) and 3,5,5-trimethyl-2-cyclopenten-2-ol-1-one (II) as the minor products. Similarly, ketimine derivative of (I) was alkylated with methyl iodide and ethyl iodide to yield the corresponding (II), (III), and 5-methyl-5-ethyl-2-cyclopenten-2-ol-1-one (VII), 3-methyl-5-ethyl-2-cyclopenten-2-ol-1-one (VIII), respectively, as the major products.     

Domain interaction sites of human lens betaB2-crystallin

betaB2-crystallin, the major component of beta-crystallin, is a dimer at low concentrations but can form oligomers under physiological conditions. The interaction domains have been speculated to be the beta-sheets, each of which is formed by two or more beta-strands. betaB2-crystallin consists of 16 beta-strands, 8 in the N-terminal domain and 8 in the C-terminal domain. Domain interaction sites may be removed by destroying the beta-strands, which can be done by site-specific mutations, substituting the beta-formers (Val, Phe, Leu) with Glu or Asn, strong beta-breakers. We have cloned the following beta-strand-deleted mutants, I20E, L34E, V54E, V60E, V73E, L97E, I109E, I124E, V144E, V152E, L162E, L165E, and V187E and their corresponding X --> Asn mutants. We also made two mutants, V46E and V129E, that were not on the beta-strand as controls. Disruption of protein-protein interactions was screened by a mammalian two-hybrid system assay. Protein-protein interactions decreased for all beta-strand-deleted mutants except I20E, L34E, and L162E mutants; this effect was not seen in the two mutant controls, V46E and V129E. The sequences around Val-54, Val-60, Val-73, and Leu-97 in the N-terminal region and Ile-109, Ile-124, Val-144, Val-152, Leu-165, and Val-187 in the C-terminal region that formed beta-strands appear to be important in dimerization. Some selected mutant proteins that showed strong (V46E and V129E) and reduced (V60E, V144E, V60N, and V144N) interactions were expressed in bacterial culture and were studied with spectroscopy and chromatography. The V60E and V144E mutants were found to be partially unfolded and incapable of forming a complete dimer.     

Overlapping LQT1 and LQT2 phenotype in a patient with long QT syndrome associated with loss-of-function variations in KCNQ1 and KCNH2

Long QT syndrome (LQTS) is an inherited disorder characterized by prolonged QT intervals and potentially life-threatening arrhythmias. Mutations in 12 different genes have been associated with LQTS. Here we describe a patient with LQTS who has a mutation in KCNQ1 as well as a polymorphism in KCNH2. The proband (MMRL0362), a 32-year-old female, exhibited multiple ventricular extrasystoles and one syncope. Her ECG (QT interval corrected for heart rate (QTc) = 518ms) showed an LQT2 morphology in leads V4-V6 and LQT1 morphology in leads V1-V2. Genomic DNA was isolated from lymphocytes. All exons and intron borders of 7 LQTS susceptibility genes were amplified and sequenced. Variations were detected predicting a novel missense mutation (V110I) in KCNQ1, as well as a common polymorphism in KCNH2 (K897T). We expressed wild-type (WT) or V110I Kv7.1 channels in CHO-K1 cells cotransfected with KCNE1 and performed patch-clamp analysis. In addition, WT or K897T Kv11.1 were also studied by patch clamp. Current-voltage (I-V) relations for V110I showed a significant reduction in both developing and tail current densities compared with WT at potentials >+20 mV (p < 0.05; n = 8 cells, each group), suggesting a reduction in IKs currents. K897T- Kv11.1 channels displayed a significantly reduced tail current density compared with WT-Kv11.1 at potentials >+10 mV. Interestingly, channel availability assessed using a triple-pulse protocol was slightly greater for K897T compared with WT (V0.5 = -53.1 ± 1.13 mV and -60.7 ± 1.15 mV for K897T and WT, respectively; p < 0.05). Comparison of the fully activated I-V revealed no difference in the rectification properties between WT and K897T channels. We report a patient with a loss-of-function mutation in KCNQ1 and a loss-of-function polymorphism in KCNH2. Our results suggest that a reduction of both IKr and IKs underlies the combined LQT1 and LQT2 phenotype observed in this patient.     

Partial covalent structure of the human alpha 2 type V collagen chain

Human cDNA libraries were screened with a cDNA fragment presumably encoding the 3' terminus of a procollagen carboxyl propeptide not identifiable as types I, II, III, or IV by protein sequence or Northern blot hybridization. One clone contained a 1350-base pair insert coding in part for 55 uninterrupted Gly-X-Y triplets. Comparison with the amino acid composition of the COOH-terminal cyanogen bromide (CB) peptides of the alpha 1 and alpha 2 type V collagen chains showed similarity only to the alpha 2(V)CB fragment. To identify the NH2 terminus of the peptide designated by methionine, an additional isolate was sequenced and found to contain a Gly-Met-Pro triplet. Thirty-one amino acids from the NH2 terminus of the alpha 2(V)CB9 fragment were then determined by Edman degradation and found to be identical to those derived from the cDNA clone. The DNA sequence encoding part of the triple helical region establishes for the first time the partial structure of a type V collagen chain. Although comparison of residues 796-1020 of the alpha 2(V) collagenous region with alpha 1 (III), alpha 1(I), and alpha 2(I) shows strong conservation of charged positions, the latter three chains appear considerably more similar to each other than to alpha 2(V). A striking feature of the alpha 2(V) sequence between 918-944 is the absence of proline residues. In the analogous region of alpha 1(I) where this amino acid is also lacking, a flexible site in the rigid triple helical structure of type I collagen has been observed (Hofmann, H., Voss, T., Kuhn, K. and Engel, J. (1984) J. Mol. Biol. 172, 325-343).     

Motif decomposition of the phosphotyrosine proteome reveals a new N-terminal binding motif for SHIP2

Advances in mass spectrometry-based proteomics have yielded a substantial mapping of the tyrosine phosphoproteome and thus provided an important step toward a systematic analysis of intracellular signaling networks in higher eukaryotes. In this study we decomposed an uncharacterized proteomics data set of 481 unique phosphotyrosine (Tyr(P)) peptides by sequence similarity to known ligands of the Src homology 2 (SH2) and the phosphotyrosine binding (PTB) domains. From 20 clusters we extracted 16 known and four new interaction motifs. Using quantitative mass spectrometry we pulled down Tyr(P)-specific binding partners for peptides corresponding to the extracted motifs. We confirmed numerous previously known interaction motifs and found 15 new interactions mediated by phosphosites not previously known to bind SH2 or PTB. Remarkably, a novel hydrophobic N-terminal motif ((L/V/I)(L/V/I)pY) was identified and validated as a binding motif for the SH2 domain-containing inositol phosphatase SHIP2. Our decomposition of the in vivo Tyr(P) proteome furthermore suggests that two-thirds of the Tyr(P) sites mediate interaction, whereas the remaining third govern processes such as enzyme activation and nucleic acid binding.     

High pressure NMR reveals a variety of fluctuating conformers in beta-lactoglobulin

High pressure 1H/15N two-dimensional NMR spectroscopy has been used to study conformational fluctuation in bovine beta-lactoglobulin at pH 2.0 and 36 degrees C. Pressure dependencies of 1H and 15N chemical shifts and cross-peak intensities were analyzed at more than 80 independent atom sites between 30 and 2000 bar. Unusually large and non-linear chemical shift pressure dependencies are found for residues centering in the hydrophobic core region, suggesting the existence of low-lying excited native states (N') of the protein. Measurement of 1H/15N cross-peak intensities at individual amide sites as a function of pressure suggests that unfolding events occur independently in two sides of the beta-barrel, i.e. the hydrophobic core side (betaF-H) (producing I2) and the non-core side (betaB-E) (producing I1). At 1 bar the stability is higher for the core region (DeltaG0 = 6.5(+/-2.0) kcal/mol) than for the non-core region (4.6(+/-1.3) kcal/mol), but at high pressure the stability is reversed due to a larger DeltaV value of unfolding for the core region (90.0(+/-35.2) ml/mol) than that for the non-core region (57.4(+/-14.4) ml/mol), possibly due to an uneven distribution of cavities. The DeltaG0 profile along the amino acid sequence obtained from the pressure experiment is found to coincide well with that estimated from hydrogen exchange experiments. Altogether, the high pressure NMR experiment has revealed a variety of fluctuating conformers of beta-lactoglobulin, notably N, N', I1, I2 and the totally unfolded conformer U. Fluctuation of N to I1 and I2 conformers with open barrel structures could be a common design of lipocalin family proteins which bind various hydrophobic compounds in its barrel structure.     

Type V collagen: molecular structure and fibrillar organization of the chicken alpha 1(V) NH2-terminal domain, a putative regulator of corneal fibrillogenesis

Previous work from our laboratories has demonstrated that: (a) the striated collagen fibrils of the corneal stroma are heterotypic structures composed of type V collagen molecules coassembled along with those of type I collagen, (b) the high content of type V collagen within the corneal collagen fibrils is one factor responsible for the small, uniform fibrillar diameter (25 nm) characteristic of this tissue, (c) the completely processed form of type V collagen found within tissues retains a large noncollagenous region, termed the NH2- terminal domain, at the amino end of its alpha 1 chain, and (d) the NH2- terminal domain may contain at least some of the information for the observed regulation of fibril diameters. In the present investigation we have employed polyclonal antibodies against the retained NH2- terminal domain of the alpha 1(V) chain for immunohistochemical studies of embryonic avian corneas and for immunoscreening a chicken cDNA library. When combined with cDNA sequencing and molecular rotary shadowing, these approaches provide information on the molecular structure of the retained NH2-terminal domain as well as how this domain might function in the regulation of fibrillar structure. In immunofluorescence and immunoelectron microscopy analyses, the antibodies against the NH2-terminal domain react with type V molecules present within mature heterotypic fibrils of the corneal stroma. Thus, epitopes within at least a portion of this domain are exposed on the fibril surface. This is in marked contrast to mAbs which we have previously characterized as being directed against epitopes located in the major triple helical domain of the type V molecule. The helical epitopes recognized by these antibodies are antigenically masked on type V molecules that have been assembled into fibrils. Sequencing of the isolated cDNA clones has provided the conceptual amino acid sequence of the entire amino end of the alpha 1(V) procollagen chain. The sequence shows the location of what appear to be potential propeptidase cleavage sites. One of these, if preferentially used during processing of the type V procollagen molecule, can provide an explanation for the retention of the NH2-terminal domain in the completely processed molecule. The sequencing data also suggest that the NH2-terminal domain consists of several regions, providing a structure which fits well with that of the completely processed type V molecule as visualized by rotary shadowing.     

Isoleucine 69 and valine 325 form a specificity pocket in human muscle creatine kinase

Creatine kinase (CK) catalyzes the reversible phosphorylation of creatine by ATP. From a structural perspective, the enzyme utilizes two flexible loop regions to sequester and position the substrates for catalysis. There has been debate over the specific roles of the flexible loops in substrate specificity and catalysis in CK and other related phosphagen kinases. In CK, two hydrophobic loop residues, I69 and V325, make contacts with the N-methyl group of creatine. In this study, we report the alteration of the substrate specificity of CK through the mutagenesis of V325. The V325 to glutamate mutation results in a more than 100-fold preference for glycocyamine, while mutation of V325 to alanine results in a slight preference of the enzyme for cyclocreatine (1-carboxymethyl-2-iminoimidazolidine). This study enhances our understanding of how the active sites of phosphagen kinases have evolved to recognize their respective substrates and catalyze their reactions.