Mária Trexler

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 β‐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.

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.

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).

NMR structure of the LCCL domain and implications for DFNA9 deafness disorder.

The LCCL domain is a recently discovered, conserved protein module named after its presence in Limulus factor C, cochlear protein Coch‐5b2 and late gestation lung protein Lgl1. The LCCL domain plays a key role in the autosomal dominant human deafness disorder DFNA9. Here we report the nuclear magnetic resonance (NMR) structure of the LCCL domain from human Coch‐5b2, where dominant mutations leading to DFNA9 deafness disorder have been identified. The fold is novel. Four of the five known DFNA9 mutations are shown to involve at least partially solvent‐exposed residues. Except for the Trp91Arg mutant, expression of these four LCCL mutants resulted in misfolded proteins. These results suggest that Trp91 participates in the interaction with a binding partner. The unexpected sensitivity of the fold with respect to mutations of solvent‐accessible residues might be attributed to interference with the folding pathway of this disulfide‐containing domain.

Both WFIKKN1 and WFIKKN2 have high affinity for growth and differentiation factors 8 and 11.

WFIKKN1 and WFIKKN2 are large extracellular multidomain proteins consisting of a WAP, a follistatin, an immunoglobulin, two Kunitz-type protease inhibitor domains, and an NTR domain. Recent experiments have shown that WFIKKN2 protein binds mature GDF8/myostatin and myostatin propeptide and inhibits the biological activity of myostatin (Hill, J. J., Qiu, Y., Hewick, R. M., and Wolfman, N. M. (2003) Mol. Endocrinol. 17, 1144–1154). Here we show that the paralogue of this protein, WFIKKN1, also binds to both myostatin and myostatin propeptide and that both WFIKKN1 and WFIKKN2 bind GDF11, the growth and differentiation factor most closely related to myostatin, with high affinity. Structure-function studies on WFIKKN1 have revealed that the follistatin domain is primarily responsible for the binding of mature growth factor, whereas the NTR domain contributes most significantly to the interaction with myostatin propeptide. Analysis of the evolutionary histories of WFIKKN1/WFIKKN2 and GDF8/GDF11 proteins indicates that the functional association of an ancestral WFIKKN protein with an ancestor of GDF8/11 may date back to cephalochordates/urochordates. Although duplication of the corresponding genes gave rise to WFIKKN1/WFIKKN2 and GDF8/GDF11 in early vertebrates, the data presented here suggest that there is significant functional overlap of the paralogous proteins.

Distinct expression pattern of two related human proteins containing multiple types of protease-inhibitory modules.

Abstract We have recently identified a gene (the WFIKKN gene) on human chromosome 16 (16p13.3) that encodes a secreted protein containing WAPtype, Follistatin/ Kazal type, Kunitztype and NTRtype proteaseinhibitory modules and an Immunoglobulin domain [Trexler et al., Proc. Natl. Acad. Sci. USA 98 (2001), 3705 3709]. In the present work we show that a gene on chromosome 17 encodes a WFIKKNrelated protein (WFIKKNRP) that has the same domain organization as the WFIKKN protein. The exonintron structure of the two genes is also similar as both genes have a single phase 0 intron that splits their WAP domains in equivalent positions. In view of the presence of several protease inhibitory modules in these proteins it seems likely that they serve to control the action of multiple types of proteases. The tissue expression pattern of the two proteins, however, is markedly different suggesting that they have distinct biological roles. Whereas the WFIKKN gene is expressed primarily in adult pancreas, liver and thymus but not in brain and ovary, significant expression of the WFIKKNRP gene is observed in ovary, testis and brain, but not in liver. Pronounced differences could also be seen in the case of fetal tissues: expression of the WFIKKN gene was highest in the lung, skeletal muscle and liver, whereas the WFIKKNRP gene was expressed primarily in brain, skeletal muscle, thymus and kidney.

A novel COCH mutation, V104del, impairs folding of the LCCL domain of cochlin and causes progressive hearing loss

Recently the causative gene of autosomal dominant sensorineural nonsyndromic late onset hearing loss (DFNA9) has been identified as the COCH gene, which lies in the DFNA9 region of human chromosome 14 (gene map locus 14q12–q13). Molecular analysis of cases of DFNA9 have identified several families with five different mutations in this gene.1–5 The cochlin protein encoded by COCH is an extracellular matrix protein that contains an LCCL domain and two von Willebrand type A domains.1 Interestingly, all mutations causing DFNA9 type deafness disorder affect the LCCL domain of cochlin. In view of the central role of this domain in the DFNA9 disorder, we initiated studies on it to explore the molecular basis of the disease.6 We found that most DFNA9 mutations affected conserved structural elements of the LCCL fold and disrupted the proper folding of this domain; recombinant mutant LCCL domains expressed in Escherichia coli failed to adopt the wild type fold and instead formed insoluble aggregates.7 It is noteworthy that insoluble deposits are detected in temporal bone sections of individuals affected by DFNA9.8 These results support the notion that the DFNA9 mutations may act through a gain of function mechanism; it is the presence of the abnormal protein that causes the disease. According to this view, accumulation of deposits in vestibular and cochlear nerve channels leads to strangulation and progressive degeneration of the dendrites, and loss of cochlear and vestibular neurones. Our present investigation was aimed at identifying novel mutations affecting the LCCL domain of cochlin in order to gain more insight into the pathomechanism of DFNA9. Hungarian patients selected according to the diagnostic criteria of DFNA9 were screened for the presence of mutations affecting the LCCL domain. These studies identified one person heterozygous for a novel mutation in the coding region of …

The solution structure of kringle 4: NMR studies on native and several chemically modified kringle 4 species of human plasminogen

Kringle 4 of human plasminogen has been studied by NMR spectroscopy to define the solution structure of the kringle‐fold and to characterize the ω‐aminocarboxylic acid binding site. Aromatic and aliphatic resonances of the NMR spectrum have been identified with the aid of spin‐decoupling and NOE procedures as well as pH‐titration and metal ion probe studies. Comparison of the NMR spectrum of kringle 4 with the spectra of various kringle 4 species chemically modified at defined positions permitted the assignment of several resonances to specific residues in the kringle 4 sequence. The NOE studies revealed that Leu45 is in close proximity of the sequentially distant Trp25/Trp61 residue pair, thus delineating a definite structural feature of the kringle‐fold. The binding of 6‐aminohexanoic acid to kringle 4 was shown to cause shifts in the resonances of several aromatic residues, including those of Trp71, suggesting that several aromatic residues may be lining the ω‐aminocarboxylic acid binding site. The binding of the ligand is competitive with the binding of lanthanide ions which reveal much detail of this site.

Peptide Ligands for the Fibronectin Type II Modules of Matrix Metalloproteinase 2 (MMP-2)

The interaction of matrix metalloproteinase 2 (MMP-2) with gelatin is mediated by three repeats homologous to fibronectin type II (FN2) modules, which are inserted in the catalytic domain in proximity of the active site. We screened a random 15-mer phage display library to identify peptides that interact with the FN2 modules of MMP-2. Interestingly, the selected peptides are not gelatin-like and do not share a common, obvious sequence motif. However, they contain a high proportion of aromatic residues. The interactions of two peptides, WHWRH0RIPLQLAAGR and THSHQWRHHQFPAPT, with constructs comprising the in-tandem first and second and second and third FN2 modules of MMP-2 (Col-12 and Col-23, respectively) were characterized by NMR. Both peptides interact with Col-12 and Col-23 with apparent association constants in the mm −1 range. Peptide binding results in perturbation of signals from residues located in the gelatin-binding pocket and flexible parts of the molecule. Although the former finding suggests that the gelatin-binding site is involved in the contact, the interpretation of the latter is less straightforward and may well reflect both the direct and indirect effects of the interaction.

Expression and characterization of the olfactomedin domain of human myocilin.

The olfactomedin-domain has been first identified in olfactomedin, an extracellular matrix protein of the olfactory neuroepithelium. Members of this extracellular domain-family have since been shown to be present in several metazoan proteins, such as latrophilins, myocilins, and noelins, but their biological function is unknown. The olfactomedin-domain of myocilin is of considerable interest, since mutations affecting this domain are associated with primary open angle glaucoma. In order to define structural features of this domain-type we have expressed the olfactomedin-domain of human myocilin in Pichia pastoris. The olfactomedin-domain contains a single disulphide-bond connecting Cys-245 and Cys-433 residues; secondary structure predictions and circular dichroism studies indicate that it consists primarily of beta-strands. It is noteworthy that the majority of mutations associated with severe forms of glaucoma affect residues that reside in conserved secondary structural elements of the olfactomedin-domain or are otherwise critical for the integrity of this protein-fold.