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M Trexler

Publications and source records attributed to M Trexler.

13 recordsLinked to original sources
PubMed2001

A human protein containing multiple types of protease-inhibitory modules.

By using sensitive homology-search and gene-finding programs, we have found that a genomic region from the tip of the short arm of human chromosome 16 (16p13.3) encodes a putative secreted protein consisting of a domain related to the whey acidic protein (WAP) domain, a domain homologous with follistatin modules of the Kazal-domain family (FS module), an immunoglobulin-related domain (Ig domain), two tandem domains related to Kunitz-type protease inhibitor modules (KU domains), and a domain belonging to the recently defined NTR-module family (NTR domain). The gene encoding these WAP, FS, Ig, KU, and NTR modules (hereafter referred to as the WFIKKN gene) is intron-depleted--its single 1,157-bp intron splits the WAP module. The validity of our gene prediction was confirmed by sequencing a WFIKKN cDNA cloned from a lung cDNA library. Studies on the tissue-expression pattern of the WFIKKN gene have shown that the gene is expressed primarily in pancreas, kidney, liver, placenta, and lung. As to the function of the WFIKKN protein, it is noteworthy that it contains FS, WAP, and KU modules, i.e., three different module types homologous with domains frequently involved in inhibition of serine proteases. The protein also contains an NTR module, a domain type implicated in inhibition of zinc metalloproteinases of the metzincin family. On the basis of its intriguing homologies, we suggest that the WFIKKN protein is a multivalent protease inhibitor that may control the action of multiple types of serine proteases as well as metalloproteinase(s).

Amino Acid Sequence↗
PubMed2001

Localization of disulfide bonds in the frizzled module of Ror1 receptor tyrosine kinase.

The frizzled (FRZ) module is a novel module type that was first identified in G-protein-coupled receptors of the frizzled and smoothened families and has since been shown to be present in several secreted frizzled-related proteins, in some modular proteases, in collagen XVIII, and in various receptor tyrosine kinases of the Ror family. The FRZ modules constitute the extracellular ligand-binding region of frizzled receptors and are known to mediate signals of WNT family members through these receptors. With an eye toward defining the structure of this important module family, we have expressed the FRZ domain of rat Ror1 receptor tyrosine kinase in Pichia pastoris. By proteolytic digestion and amino acid sequencing the disulfide bonds were found to connect the 10 conserved cysteines in a 1-5, 2-4, 3-8, 6-10, and 7-9 pattern. Circular dichroism and differential scanning calorimetry studies on the recombinant protein indicate that the disulfide-bonded FRZ module corresponds to a single, compact, and remarkably stable folding domain possessing both alpha-helices and beta-strands.

Amino Acid Sequence↗
PubMed2000

Worldwide DNA sequence variation in a 10-kilobase noncoding region on human chromosome 22.

Human DNA sequence variation data are useful for studying the origin, evolution, and demographic history of modern humans and the mechanisms of maintenance of genetic variability in human populations, and for detecting linkage association of disease. Here, we report worldwide variation data from a approximately 10-kilobase noncoding autosomal region. We identified 75 variant sites in 64 humans (128 sequences) and 463 variant sites among the human, chimpanzee, and orangutan sequences. Statistical tests suggested that the region is selectively neutral. The average nucleotide diversity (pi) across the region was 0.088% among all of the human sequences obtained, 0.085% among African sequences, and 0.082% among non-African sequences, supporting the view of a low nucleotide diversity ( approximately 0.1%) in humans. The comparable pi value in non-Africans to that in Africans indicates no severe bottleneck during the evolution of modern non-Africans; however, the possibility of a mild bottleneck cannot be excluded because non-Africans showed considerably fewer variants than Africans. The present and two previous large data sets all show a strong excess of low frequency variants in comparison to that expected from an equilibrium population, indicating a relatively recent population expansion. The mutation rate was estimated to be 1.15 x 10(-9) per nucleotide per year. Estimates of the long-term effective population size N(e) by various statistical methods were similar to those in other studies. The age of the most recent common ancestor was estimated to be approximately 1.29 million years ago among all of the sequences obtained and approximately 634,000 years ago among the non-African sequences, providing the first evidence from a noncoding autosomal region for ancient human histories, even among non-Africans.

Animals↗
PubMed2000

The LCCL module.

Here we show that Lgl1 protein, cub-1-related proteins, coch-5b2-related proteins, coagulation factor C of horse-shoe crab and a predicted protein of Plasmodium falciparum share a homologous domain. Since this domain-type was first identified in Limulus factor C, Coch-5b2 and Lgl1 we propose the name LCCL for this domain-family. The LCCL module of coch-5b2 is of special biological interest because it has been shown recently that mutations affecting this module cause the deafness disorder DFNA9 in humans. With a view to defining the structure and function of the LCCL domain of human coch-5b2 protein, we have expressed it in Escherichia coli and subjected it to preliminary structural characterization. Structure prediction and circular dichroism studies on the recombinant protein indicate that the domain possesses both alpha helices and beta strands. It is shown that the mutations which cause hearing loss in humans affect residues that are critical for the integrity of the LCCL module of the coch-5b2 protein.

Amino Acid Sequence↗
PubMed1992

Refined solution structure and ligand-binding properties of PDC-109 domain b. A collagen-binding type II domain.

We have determined, via 1H-n.m.r., the solution conformation of the collagen-binding b-domain of the bovine seminal fluid protein PDC-109 (PDC-109/b). The structure determination is based on 341 interproton distance estimates and 42 dihedral angle estimates: a set of 24 initial structures were computed; 12 using the variable target function program DIANA, and 12 using the metric matrix program DISGEO. These structures were optimized by restrained energy minimization and dynamic simulated annealing using the CHARMM and X-PLOR programs. The average pairwise root-mean-square difference (r.m.s.d) between the optimized DIANA (DISGEO) structures is 0.71 A (0.82 A) for the backbone atoms, and 1.73 A (2.03 A) for all atoms. Both sets of structures exhibit the same global fold, secondary structure and placement of most non-polar side-chains. Two central antiparallel beta-sheets, which lie roughly perpendicular to each other, and two irregular loops support a large, partially exposed, hydrophobic surface that defines a putative binding site. A test of a hybrid relaxation matrix-based distance refinement protocol (MIDGE program) was performed using a normalized 250 millisecond NOESY spectrum. The resulting distances were input to the molecular mechanics/dynamics procedures mentioned above in order to optimize the DIANA structures. Our results indicate that relaxation matrix refinement of distances is most useful when used conservatively for identifying underestimated distance constraints. 1H-n.m.r. monitored ligand titration experiments revealed definite, albeit weak, binding interactions for phenethylamine and leucine analogs (Ka less than or equal to 25 M-1). Residues perturbed by ligand binding include Tyr7, Trp26, Tyr33, Asp34 and Trp39. These results suggest that PDC-109/b may recognize specific leucine and/or isoleucine-containing sequences within collagen.

Amino Acid Sequence↗
PubMed1991

Sequence-specific 1H NMR assignments and structural characterization of bovine seminal fluid protein PDC-109 domain b.

Sequence-specific resonance assignments for the isolated second or b domain of the bovine seminal fluid protein PDC-109 have been obtained from analysis of two-dimensional 1H NMR experiments recorded at 500 MHz. These assignments include the identification of all aromatic and most aliphatic amino acid resonances. Stereospecific assignment of resonances stemming from the Val2 CH3 gamma,gamma' groups and from seven CH beta,beta' geminal pairs has been accomplished by analysis of 3J alpha beta coupling constants in conjunction with patterns of cross-peak intensities observed in two-dimensional nuclear Overhauser effect (NOESY) spectra. Analysis of NOESY and 3J alpha NH data reveals a small antiparallel beta-sheet involving stretches containing residues 25-28 and 39-42, a cis-proline residue (Pro4), antiparallel strands consisting of residues 1-3, 5-7, and 10-13, and an aromatic cluster composed of Tyr7, Trp26, and Tyr33. The results of distance geometry and restrained molecular dynamics calculations indicate that the global fold of the PDC-109 b domain, a type II module related to those found in fibronectin, is somewhat different from that predicted by modeling the structure on the basis of homology between type II and kringle units. A shallow depression in the molecular surface which presents a solvent-exposed hydrophobic area--a potential ligand-binding site-is identified in the NMR-based models.

Amino Acid Sequence↗
PubMed1990

The collagen-binding site of type-II units of bovine seminal fluid protein PDC-109 and fibronectin.

A single type-II domain has been isolated by limited proteolysis of the collagen-binding bovine seminal fluid protein, PDC-109. The 45-residue fragment corresponding to the second type-II domain of the parent molecule was found to have retained affinity for immobilized collagen, indicating that this minidomain carries critical regions of the collagen-binding site. Studies on various fragments of fibronectin have also implicated the two type-II units of this molecule in collagen-binding. In the present work we have found that type-II domains of human fibronectin, expressed in Escherichia coli as beta-galactosidase fusion proteins, bind specifically to immobilized collagen.

Amino Acid Sequence↗
PubMed1985

Chemical modification and nuclear magnetic resonance studies on human plasminogen kringle 4. Assignment of tyrosine and histidine resonances to specific residues in the sequence.

Modification of kringle 4 with tetranitromethane leads to the selective nitration of tyrosine 40 but on prolonged incubation with reagent, reaction of tyrosine 49 is also observed. Nitration of tyrosines 40 and 49 had no influence on the lysine-Sepharose affinity of kringle 4, indicating that these residues are not important for the functional integrity of the ligand-binding site. Comparison of the NMR spectra of native kringle 4 with those of kringle 4 in which tyrosine 40 or tyrosines 40 and 49 are nitrated permitted the identification of the resonances of these residues. These NMR studies also showed that the chemical modifications caused little perturbation of the three-dimensional structure of the protein. Cross-linking of lysine 35 and tyrosine 40 with 1,3-difluoro-4,6-dinitrobenzene demonstrates that in the kringle-fold the reactive epsilon-amino and phenolic groups of these residues can approach each other to a distance of 0.5 nm. NMR spectra of this kringle 4 species also confirmed the assignment of the resonances to tyrosine 40. NMR spectra of a kringle 4 derivative in which the disulphide bridge between cysteines 1 and 79 has been broken by selective reduction and alkylation showed that the core structure of the kringle-fold and the lysine-binding site are unaltered by this modification. This observation is in agreement with earlier results which showed that the lysine-Sepharose affinity of kringle 4 is not affected by reduction and alkylation of this disulphide bridge. Comparison of the NMR spectra of native and disulphide-cleaved kringle 4 aided in the assignment of resonances to residues adjacent to the site of modification (tyrosine 2 and histidine 3) and permitted the tentative assignment of the resonances of tyrosines 9 and 73.

Amino Acids↗
PubMed1985

Analysis and identification of aromatic signals in the proton magnetic resonance spectrum of the kringle 4 fragment from human plasminogen.

The aromatic 1H NMR spectrum of the kringle 4 domain from human plasminogen has been reexamined in order to identify signals stemming from individual residues. Acid-base titration, nuclear Overhauser effect experiments, and two-dimensional correlated spectroscopies have been implemented in order to analyze the spectrum both in the presence and in the absence of ligands. All six histidyl imidazole singlets have been recognized and paired according to their common side-chain origin. A similar identification has been achieved for the three sets of tryptophanyl resonances, and for Trp-I, the correspondence between indole singlet and multiplets is unambiguously established. The single phenylalanyl side chain and all tyrosyl phenol spin systems have been identified. Titration experiments indicate that one or two of the tryptophans are in the vicinity of carboxyl groups. It is shown that the spectrum for one tyrosyl ring, Tyr-V, undetectable at approximately 300 MHz, becomes visible at 600 MHz, reflecting slow motion on the NMR time scale and a constrained location within the kringle. A simulation of the complete kringle 4 aromatic spectrum is included.

Humans↗
PubMed1984

Residues Cys-1 and Cys-79 are not essential for refolding of reduced-denatured kringle 4 fragment of human plasminogen.

It was shown previously that the Cys-1-Cys-79 disulphide bond forms in the last step of refolding of kringle 4 and that this bond is not essential for the lysine-Sepharose affinity of the kringle 4 fragment (Trexler, M. and Patthy, L. (1983) Proc. Natl. Acad. Sci. U.S.A. 80 2457-2461). Here we show that kringle 4, carboxymethylated on Cys-1 and Cys-79, regains its lysine-Sepharose affinity following denaturation and reductive cleavage of its disulphide bonds. The rate of refolding under aerobic conditions or in the presence of oxidized and reduced glutathione was similar to that observed in the case of native kringle 4. Our results suggest that Cys-1 and Cys-79 residues of kringles are not essential for the maintenance or acquisition of the biologically active kringle-fold.

Amino Acids↗
PubMed1984

Kringles: modules specialized for protein binding. Homology of the gelatin-binding region of fibronectin with the kringle structures of proteases.

Prothrombin, plasminogen, urokinase- and tissue-type plasminogen activators contain homologous structures known as kringles . The kringles correspond to autonomous structural and folding domains which mediate the binding of these multidomain proteins to other proteins. During evolution the different kringles retained the same gross architecture, the kringle -fold, yet diverged to bind different proteins. We show that the amino acid sequences of the type II structures of the gelatin-binding region of fibronectin are homologous with those of the protease- kringles . Prediction of secondary structures revealed a remarkable agreement in the positions of predicted beta-sheets, suggesting that the folding of kringles and type II structures may also be similar. As a corollary of this finding, the disulphide-bridge pattern of type II structures is shown to be homologous to that in kringles . It is noteworthy that protease- kringles and fibronectin type II structures have similar functions inasmuch as they mediate the binding of multidomain proteins to other proteins. It is proposed that the kringles of proteases and type II structures of fibronectin evolved from a common ancestral protein binding module.

Animals↗
PubMed1983

Folding autonomy of the kringle 4 fragment of human plasminogen.

Kringle 4, an 88-residue plasminogen fragment carrying a lysine-binding site, loses its affinity for lysine-Sepharose upon reductive cleavage of its disulfide bridges. Aerobic incubation of the reduced, denatured fragment results in the rapid restoration of the disulfide bonds with concomitant recovery of lysine-Sepharose affinity. The ability of the unfolded fragment to regain its native conformation suggests that the kringle structure is an autonomous folding domain. During refolding of kringle 4 the native disulfide bonds, (formula; see text) and (formula; see text), appears first. The folding intermediate possessing these two disulfide bridges already binds to lysine-Sepharose, indicating that the third native bridge, which in native kringle 4 connects residues Cys1 and Cys79, is not essential for the maintenance of the biologically active conformation of kringle 4. Comparison of the sequences of human prothrombin, urokinase, and plasminogen kringles revealed that the residues surrounding the (formula; see text) and (formula; see text) bridges constitute the most conservative segments of kringles, whereas the residues neighboring the (formula; see text) bridge are not highly conserved. We propose that conservation of various residues in the different kringles reflects their importance for the folding autonomy of kringles.

Amino Acid Sequence↗