Marian Seto

Structural basis for the anticoagulant activity of the thrombin-thrombomodulin complex.

The serine proteinase α-thrombin causes blood clotting through proteolytic cleavage of fibrinogen and protease-activated receptors and amplifies its own generation by activating the essential clotting factors V and VIII. Thrombomodulin, a transmembrane thrombin receptor with six contiguous epidermal growth factor-like domains (TME1–6), profoundly alters the substrate specificity of thrombin from pro- to anticoagulant by activating protein C (see, for example, reference 2). Activated protein C then deactivates the coagulation cascade by degrading activated factors V and VIII. The thrombin–thrombomodulin complex inhibits fibrinolysis by activating the procarboxypeptidase thrombin-activatable fibrinolysis inhibitor. Here we present the 2.3 Å crystal structure of human α-thrombin bound to the smallest thrombomodulin fragment required for full protein-C co-factor activity, TME456. The Y-shaped thrombomodulin fragment binds to thrombins anion-binding exosite-I, preventing binding of procoagulant substrates. Thrombomodulin binding does not seem to induce marked allosteric structural rearrangements at the thrombin active site. Rather, docking of a protein C model to thrombin–TME456 indicates that TME45 may bind substrates in such a manner that their zymogen-activation cleavage sites are presented optimally to the unaltered thrombin active site.

Corin, a Mosaic Transmembrane Serine Protease Encoded by a Novel cDNA from Human Heart

A novel cDNA has been identified from human heart that encodes an unusual mosaic serine protease, designated corin. Corin has a predicted structure of a type II transmembrane protein and contains two frizzled-like cysteine-rich motifs, seven low density lipoprotein receptor repeats, a macrophage scavenger receptor-like domain, and a trypsin-like protease domain in the extracellular region. Northern analysis showed that corin mRNA was highly expressed in the human heart. In mice, corin mRNA was detected by in situ hybridization in the cardiac myocytes of the embryonic heart as early as embryonic day (E) 9.5. By E11.5–13.5, corin mRNA was most abundant in the primary atrial septum and the trabecular ventricular compartment. Expression in the heart was maintained through the adult. In addition, mouse corin mRNA was also detected in the prehypertrophic chrondrocytes in developing bones. By fluorescentin situ hybridization analysis, the human corin gene was mapped to 4p12–13 where a congenital heart disease locus, total anomalous pulmonary venous return, had been previously localized. The unique domain structure and specific embryonic expression pattern suggest that corin may have a function in cell differentiation during development. The chromosomal localization of the human corin gene makes it an attractive candidate gene for total anomalous pulmonary venous return.

Protein fold analysis of the B30.2-like domain.

The B30.2‐like domain occurs in some members of a diverse and growing family of proteins containing zinc‐binding B‐box motifs, whose functions include regulation of cell growth and differentiation. The B30.2‐like domain is also found in proteins without the zinc‐binding motifs, such as butyrophilin (a transmembrane glycoprotein) and stonustoxin (a secreted cytolytic toxin). Currently, the function for the B30.2‐like domain is not clear and the structure of a protein containing this domain has not been solved. The secondary structure prediction methods indicate that the B30.2‐like domain consists of fifteen or fewer β‐strands. Fold recognition methods identified different structural topologies for the B30.2‐like domains. Secondary structure prediction, deletion and lack of local sequence identity at the C‐terminal region for certain members of the family, and packing of known core structures suggest that a structure containing two beta domains is the most probable of these folds. The most C‐terminal sequence motif predicted to be a β‐strand in all B30.2‐like domains is a potential subdomain boundary based on the sequence‐structure alignments. Models of the B30.2‐like domains were built based on immunoglobulin‐like folds identified by the fold recognition methods to evaluate the possibility of the B30.2 domain adopting known folds and infer putative functional sites. The SPRY domain has been identified as a subdomain within the B30.2‐like domain. If the B30.2‐like domain is a subclass of the SPRY domain family, then this analysis would suggest that the SPRY domains are members of the immunoglobulin superfamily. Proteins 1999;35:235–249.

The Structure of a 19-Residue Fragment from the C-loop of the Fourth Epidermal Growth Factor-like Domain of Thrombomodulin

The solution structure has been determined for a 19-residue peptide that is fully folded at room temperature. The sequence of this peptide is based on the C-loop, residues 371-389, of the fourth epidermal growth factor-like domain of thrombomodulin, a protein that acts as a cofactor for the thrombin activation of protein C. Despite its small size, the peptide forms a compact structure with almost no repeating secondary structure. The results indicate the structure is held together by hydrophobic interactions, which in turn stabilize the two β-turns in the structure. The first β-turn in the C-loop represents a conserved motif that is found in the published structures of five other epidermal growth factor-like proteins. The critical role of Phe376 in the stabilization of the first β-turn is consistent with mutagenesis data with soluble thrombomodulin. The results also show that a small subdomain of a larger protein can fold independently, and therefore it could act as an initiation site for further folding.

A model of the acid sphingomyelinase phosphoesterase domain based on its remote structural homolog purple acid phosphatase

Sequence profile and fold recognition methods identified mammalian purple acid phosphatase (PAP), a member of a dimetal‐containing phosphoesterase (DMP) family, as a remote homolog of human acid sphingomyelinase (ASM). A model of the phosphoesterase domain of ASM was built based on its predicted secondary structure and the metal‐coordinating residues of PAP. Due to the low sequence identity between ASM and PAP (∼15%), the highest degree of confidence in the model resides in the metal‐binding motifs. The ASM model predicts residues Asp 206, Asp 278, Asn 318, His 425, and His 457 to be dimetal coordinating. A putative orientation for the phosphorylcholine head group of the ASM substrate, sphingomyelin (SM), was made based on the predicted catalysis of the phosphorus–oxygen bond in the active site of ASM and on a structural comparison of the PAP–phosphate complex to the C‐reactive protein–phosphorylcholine complex. These complexes revealed similar spatial interactions between the metal‐coordinating residues, the metals, and the phosphate groups, suggesting a putative orientation for the head group in ASM consistent with the mechanism considerations. A conserved sequence motif in ASM, NX3CX3N, was identified (Asn 381 to Asn 389) and is predicted to interact with the choline amine moiety in SM. The resulting ASM model suggests that the enzyme uses an SN2‐type catalytic mechanism to hydrolyze SM, similar to other DMPs. His 319 in ASM is predicted to protonate the ceramide‐leaving group in the catalysis of SM. The putative functional roles of several ASM Niemann‐Pick missense mutations, located in the predicted phosphoesterase domain, are discussed in context to the model.

The Roles of Selected Arginine and Lysine Residues of TAFI (Pro-CPU) in Its Activation to TAFIa by the Thrombin-Thrombomodulin Complex

Thrombomodulin (TM) increases the catalytic efficiency of thrombin (IIa)-mediated activation of thrombin-activable fibrinolysis inhibitor (TAFI) 1250-fold. Negatively charged residues of the C-loop of TM-EGF-like domain 3 are required for TAFI activation. Molecular models suggested several positively charged residues of TAFI with which the C-loop residues could interact. Seven TAFI mutants were constructed to determine if these residues are required for efficient TAFI activation. TAFI wild-type or mutants were activated in the presence or absence of TM and the kinetic parameters of TAFI activation were determined. When the three consecutive lysine residues in the activation peptide of TAFI were substituted with alanine (K42/43/44A), the catalytic efficiencies for TAFI activation with TM decreased 8-fold. When other positively charged surface residues of TAFI (Lys-133, Lys-211, Lys-212, Arg-220, Lys-240, or Arg-275) were mutated to alanine, the catalytic efficiencies for TAFI activation with TM decreased by 1.7–2.7-fold. All decreases were highly statistically significant. In the absence of TM, catalytic efficiencies ranged from 2.8-fold lower to 1.24-fold higher than wild-type. None of these, except the 2.8-fold lower value, was statistically significant. The average half-life of the TAFIa mutants was 8.1 ± 0.6 min, and that of wild type was 8.4 ± 0.3 min at 37 °C. Our data show that these residues are important in the activation of TAFI by IIa, especially in the presence of TM. Whether the mutated residues promote a TAFI-TM or TAFI-IIa interaction remains to be determined. In addition, these residues do not influence spontaneous inactivation of TAFIa.

Crystallographic analysis of potent and selective factor Xa inhibitors complexed to bovine trypsin.

Factor Xa is a serine protease which activates thrombin (factor IIa) and plays a key regulatory role in the blood-coagulation cascade. Factor Xa is, therefore, an important target for the design of anti-thrombotics. Both factor Xa and thrombin share sequence and structural homology with trypsin. As part of a factor Xa inhibitor-design program, a number of factor Xa inhibitors were crystallographically studied complexed to bovine trypsin. The structures of one diaryl benzimidazole, one diaryl carbazole and three diaryloxypyridines are described. All five compounds bind to trypsin in an extended conformation, with an amidinoaryl group in the S1 pocket and a second basic/hydrophobic moiety bound in the S4 pocket. These binding modes all bear a resemblance to the reported binding mode of DX-9065a in bovine trypsin and human factor Xa.