PDBsum entry 1rfn

Coagulation factor

PDB id

1rfn

Contents

			Protein chains

					235 a.a. *


					57 a.a. *

			Ligands

					PBZ


					TBU

			Metals

					_CA

			Waters ×16

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Coagulation factor ixa: the relaxed conformation of tyr99 blocks substrate binding.

Authors

K.P.Hopfner, A.Lang, A.Karcher, K.Sichler, E.Kopetzki, H.Brandstetter, R.Huber, W.Bode, R.A.Engh.

Ref.

Structure, 1999, 7, 989-996. [DOI no: 10.1016/S0969-2126(99)80125-7]

PubMed id

10467148

Abstract

BACKGROUND: Among the S1 family of serine proteinases, the blood coagulation factor IXa (fIXa) is uniquely inefficient against synthetic peptide substrates. Mutagenesis studies show that a loop of residues at the S2-S4 substrate-binding cleft (the 99-loop) contributes to the low efficiency. The crystal structure of porcine fIXa in complex with the inhibitor D-Phe-Pro-Arg-chloromethylketone (PPACK) was unable to directly clarify the role of the 99-loop, as the doubly covalent inhibitor induced an active conformation of fIXa. RESULTS: The crystal structure of a recombinant two-domain construct of human fIXa in complex with p-aminobenzamidine shows that the Tyr99 sidechain adopts an atypical conformation in the absence of substrate interactions. In this conformation, the hydroxyl group occupies the volume corresponding to the mainchain of a canonically bound substrate P2 residue. To accommodate substrate binding, Tyr99 must adopt a higher energy conformation that creates the S2 pocket and restricts the S4 pocket, as in fIXa-PPACK. The energy cost may contribute significantly to the poor K(M) values of fIXa for chromogenic substrates. In homologs, such as factor Xa and tissue plasminogen activator, the different conformation of the 99-loop leaves Tyr99 in low-energy conformations in both bound and unbound states. CONCLUSIONS: Molecular recognition of substrates by fIXa seems to be determined by the action of the 99-loop on Tyr99. This is in contrast to other coagulation enzymes where, in general, the chemical nature of residue 99 determines molecular recognition in S2 and S3-S4. This dominant role on substrate interaction suggests that the 99-loop may be rearranged in the physiological fX activation complex of fIXa, fVIIIa, and fX.



	Figure 3. Figure 3. Stereoview comparisons of substrate recognition by fIXa and other homologs. (a) The divergent structures of the fIXa 99-loop (yellow) compared with those of factor Xa (white) and tPA (red) place Tyr99 either in a relaxed position that blocks substrate binding in the S2 site (thick lines) or in a strained position that blocks the S4 site (thin lines). The inhibitor (purple, Image -Phe-Pro-Arg with proline atoms omitted for clarity) position is taken from the superposition of porcine fIXa. (b) The conformation of the fIXa 99-loop (yellow) is stabilized by a hydrogen bond between Tyr94 (phenylalanine in fXa) and Lys98, whereas the fXa 99-loop (gray) conformation is prevented by the position of Tyr177 (threonine in fXa).

PROCHECK

	Headers