The Polymerization Pocket “a” within the Carboxyl-terminal Region of the γ Chain of Human Fibrinogen Is Adjacent to but Independent from the Calcium-binding Site

Abstract

The carboxyl-terminal region of the ␥ chain of fibrinogen is involved in calcium binding, fibrin polymerization, factor XIIIa-mediated cross-linking, and binding to the platelet fibrin(ogen) receptor. to Leu 427 (␥C30) from the carboxyl end of the ␥ or ␥ chains, respectively, of human fibrinogen were expressed in yeast (Pichia pastoris) and characterized as to their cross-linking by factor XIIIa, polymerization pocket, and calcium-binding site. rFbg␥C30 and ␥C30 were both readily cross-linked by factor XIIIa, but only rFbg␥C30 was capable of inhibiting thrombin-induced platelet aggregation. Two mutants, ␥C30-Q329R and ␥C30-D364A, which were based on the three-dimensional structure of the polymerization pocket within rFbg␥C30 and on information derived from naturally occurring mutant fibrinogens, were also expressed and characterized. rFbg␥C30 inhibited (desAA)fibrin polymerization in a dose-dependent manner, while the two mutant forms did not. Similarly, rFbg␥C30 and ␥C30 were protected from plasmin degradation by the presence of Ca 2؉ or the peptide Gly-Pro-Arg-Pro, indicating that a functional Ca 2؉ -binding site and polymerization pocket are contained within each of these fragments. The mutant fragments, however, were protected from plasmin only by metal ions, while no protective effect was conferred by GPRP or by any other peptide tested. These results indicate that the polymerization pocket "a", which binds the peptide GPRP, functions independently from the nearby calcium-binding site and that amino acids Gln 329 and Asp 364 play a crucial role in fibrin polymerization.Fibrinogen is a large glycoprotein composed of six polypeptide chains (␣, ␤, ␥) 2 held together by disulfide bonds. By electron microscopy, the molecule appears as a trinodular structure (1) in which the central nodule contains the amino termini of all six chains (2). The two distal nodules contain the carboxyl-terminal regions of the ␥ and ␤ chains. Upon cleavage by thrombin, fibrinopeptides A and B are released from the amino termini of the ␣ and ␤ chains, resulting in fibrin monomers that polymerize spontaneously. The newly exposed amino termini constitute polymerization sites A and B that are complementary to polymerization pockets "a" and "b", respectively (3-6). The "a" pocket is located within the carboxyl region of the ␥ chain (7, 8), while the location of the "b" pocket remains controversial. It has been proposed to arise upon the alignment of the D domains of two fibrin molecules (6), to be contained in the carboxyl-terminal region of the ␤ chain (9, 10), and to involve the carboxyl-terminal region of the ␣ chain (11).Calcium promotes the polymerization of fibrin monomers (12-15) and the cross-linking of fibrin by factor XIIIa (16). It also protects fibrinogen fragment D against plasmic degradation (17). Fibrinogen binds three calcium ions per molecule (18,19), including one in the carboxyl-terminal region of each ␥ chain. The third calcium is present in the central nodule, provided that the ␣ chains are intact (20). The precise loc...

A double-headed Gly-Pro-Arg-Pro ligand mimics the functions of the E domain of fibrin for promoting the end-to-end crosslinking of γ chains by factor XIII a

“…Crosslinked fibrinogens larger than pentamers are marked collectively as n in Fig. 4, but, on the gel relating to the reaction in the presence of 10 M ligand, even bands representing 15 Crosslinking in the presence of Ca 2ϩ (2 mM) by activated factor XIII (0.02 M) was allowed to proceed at 37°C for 30 min when it was terminated by admixture of EDTA (4 mM). After reduction of the samples with DTT, SDS͞PAGE was performed (applying ca.…”

“…Gly-Pro-Arg-Pro, which resembles the natural Gly-Pro-Arg-Val sequences (knobs) in the fibrinopeptide A-denuded central E domain of fibrin, can prevent clot formation (10). The tetrapeptide blocks the polymerization pockets or holes located in the ␥ chains at the two D end domains of the protein (11)(12)(13)(14)(15)(16). It is important to bear in mind that these polymerization pockets are present not only in fibrin but also in the parent fibrinogen molecule, i.e., thrombin action is not required for opening them for Gly-ProArg-Pro to bind.…”

Hereditary Disorders of Fibrinogen

“…The interaction of these clusters of positive and negative charges should play an important role in the alignment of the rod‐like fibrin monomers into half‐molecule overlapping protofibrils. In fact, this hypothesis has recently been confirmed by crystallographic analyses reported independently from two laboratories 9–13. When bound to its complementary a site located on the D domain of another molecule, the α‐amino group of α Gly‐1 (Aα Gly‐17) is situated between the side chain carboxyl groups of γ Asp‐330 and γ Asp‐364.…”

Conformational preference of ChaK1 binding peptides: a molecular dynamics study

“…The first two APKs in the so called "alpha-kinases" family [2,3] were myosin heavy chain kinase A (MHCK A) from Dictyostelium [4,5], and elongation factor 2 kinase (eEF-2 kinase [6]). These kinases are involved in the regulation of a wide range of different processes, including protein translation (eEF-2 kinase [7]), myosin association (MHCK [8]), ion channel regulation (TRPM6/ChaK2, TRPM7/ChaK1 [9,10]), and cardiomyocyte differentiation (Midori [11]).…”