The use of sequence-specific antibodies to identify a secondary binding site in thrombin.

Noé, G.; Hofsteenge, Jan; Rovelli, Giorgio; Stone, Stuart R.

doi:10.1016/s0021-9258(18)37844-x

Cited by 70 publications

(7 citation statements)

References 37 publications

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“…Previous studies have shown that the tight interaction between hirudin and thrombin involves a number of different areas on both the inhibitor and the protease (Chang, 1983;Stone et al, 1987;Noé et al, 1988;Braun et al, 1988). The C-terminal region of hirudin has been shown to be important for its interactions with thrombin (Chang, 1983; Braun et al, 1988;Stone et al, 1989).…”

Section: Discussionmentioning

confidence: 99%

“…of the interaction of thrombin with hirudin does not arise solely from the binding of hirudin to the catalytic site of thrombin but that secondary sites on both molecules are also involved (Chang, 1983;Stone et al, 1987;Noé et al, 1988). The availability of recombinant hirudin (Fortkamp et al, 1986;Harvey et al, 1986;Meyhack et al, 1987;Bergmann et al, 1986; Loison et al, 1988) offers the possibility of identifying interaction sites on hirudin by site-directed mutagenesis.…”

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confidence: 99%

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Contribution of the N-terminal region of hirudin to its interaction with thrombin

Wallace¹,

Dennis²,

Hofsteenge³

et al. 1989

Biochemistry

103

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Hirudin is a 65-residue polypeptide that specifically inhibits thrombin by forming a tight, noncovalent complex with the enzyme. The role of the two amino-terminal valine residues and the N-terminal alpha-amino group of hirudin in the formation of the complex has been investigated by site-directed mutagenesis and chemical modification. Replacement of the two N-terminal valyl residues of recombinant hirudin by polar amino acids resulted in an increase in the inhibition constant (KI). In contrast, replacement of these residues by hydrophobic amino acids had little effect on the value for KI. These results demonstrated that the hydrophobic nature of the N-terminal residues of hirudin was important for its interaction with thrombin. Addition of a single amino acid to the N-terminus of hirudin resulted in a marked increase in the value of KI. A similar effect was observed when the positive charge of the alpha-amino group was removed by acetylation. In contrast, amidination of this group, which preserves the positive charge, resulted in a less pronounced increase in the value of KI. Thus, it appears that a positive charge immediately adjacent to the N-terminal hydrophobic residue is required for optimal binding to thrombin.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Contribution of the N-terminal region of hirudin to its interaction with thrombin

Wallace¹,

Dennis²,

Hofsteenge³

et al. 1989

Biochemistry

103

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“…The acidic C-terminal region of hirudin has been implicated in interactions with a secondary binding site on thrombin (Chang, 1983;Braun et al, 1988a). This negatively charged region of hirudin is thought to interact with an op-0006-2960/89/0428-6857S01.50/0 © 1989 American Chemical Society positely charged secondary binding site on thrombin (Fenton et al, 1988;Fenton & Bing, 1986; Noé et al, 1988;Stone et al, 1987). The proposed secondary binding site includes residues 62-73 of the B chain of human -thrombin, and these residues correspond to a surface loop in a-chymotrypsin (Birktoft & Blow, 1972;Furie et al, 1982).…”

mentioning

confidence: 99%

“…Cleavage within this region with trypsin produces human /3x-thrombin (Braun et al, 1988b) which has a much reduced affinity for hirudin (Stone et al, 1987). Moreover, antibodies that bind to residues 62-73 block the interaction of hirudin with thrombin (Noé et al, 1988). Evidence that the C-terminal region of hirudin binds to this secondary binding site is provided by studies with fragments of hirudin.…”

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confidence: 99%

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Quantitative evaluation of the contribution of ionic interactions to the formation of the thrombin-hirudin complex

Stone

Dennis²,

Hofsteenge³

1989

Biochemistry

129

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The effect of ionic strength on the kinetics of inhibition of human alpha-thrombin has been examined by using genetically engineered forms of hirudin that differed only in the number of negatively charged residues in the carboxyl-terminal region of the molecule. Analysis of the data obtained allowed the binding energy for the thrombin-hirudin complex to be divided into contributions from ionic and nonionic interactions. The contribution of nonionic interactions to the binding energy was the same for each of the forms whereas the ionic contribution varied with the charge of the molecule. Each of the negatively charged residues made an approximately equal contribution of -4kJ mol-1 to the binding energy. For native hirudin, ionic interactions accounted for 32% of the binding energy at an ionic strength of zero.

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The refined 1.9 A crystal structure of human alpha-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment.

et al. 1989

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A stoichiometric complex formed between human alpha‐thrombin and D‐Phe‐Pro‐Arg chloromethylketone was crystallized in an orthorhombic crystal form. Orientation and position of a starting model derived from homologous modelling were determined by Patterson search methods. The thrombin model was completed in a cyclic modelling‐crystallographic refinement procedure to a final R‐value of 0.171 for X‐ray data to 1.92 A. The structure is in full agreement with published cDNA sequence data. The A‐chain, ordered only in its central part, is positioned along the molecular surface opposite to the active site. The B‐chain exhibits the characteristic polypeptide fold of trypsin‐like proteinases. Several extended insertions form, however, large protuberances; most important for interaction with macromolecular substrates is the characteristic thrombin loop around Tyr60A‐Pro60B‐Pro60C‐Trp60D (chymotrypsinogen numbering) and the enlarged loop around the unique Trp148. The former considerably restricts the active site cleft and seems likely to be responsible for poor binding of most natural proteinase inhibitors to thrombin. The exceptional specificity of D‐Phe‐Pro‐Arg chloromethylketone can be explained by a hydrophobic cage formed by Ile174, Trp215, Leu99, His57, Tyr60A and Trp60D. The narrow active site cleft, with a more polar base and hydrophobic rims, extends towards the arginine‐rich surface of loop Lys70‐Glu80 that probably represents part of the anionic binding region for hirudin and fibrinogen.

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The use of sequence-specific antibodies to identify a secondary binding site in thrombin.

Cited by 70 publications

References 37 publications

Contribution of the N-terminal region of hirudin to its interaction with thrombin

Contribution of the N-terminal region of hirudin to its interaction with thrombin

Quantitative evaluation of the contribution of ionic interactions to the formation of the thrombin-hirudin complex

The refined 1.9 A crystal structure of human alpha-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment.

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