PDBsum entry 1tcp

Blood coagulation inhibitor

PDB id

1tcp

Contents

			Protein chain

					60 a.a.

References listed in PDB file

Key reference

Title

Nmr structure determination of tick anticoagulant peptide (tap).

Authors

M.S.Lim-Wilby, K.Hallenga, M.De maeyer, I.Lasters, G.P.Vlasuk, T.K.Brunck.

Ref.

Protein Sci, 1995, 4, 178-186. [DOI no: 10.1002/pro.5560040205]

PubMed id

7538849

Note In the PDB file this reference is annotated as "TO BE PUBLISHED". The citation details given above were identified by an automated search of PubMed on title and author names, giving a perfect match.

Abstract

Tick anticoagulant peptide (TAP) is a potent and selective 60-amino acid inhibitor of the serine protease Factor Xa (fXa), the penultimate enzyme in the blood coagulation cascade. The structural features of TAP responsible for its remarkable specificity for fXa are unknown, but the binding to its target appears to be unique. The elucidation of the TAP structure may facilitate our understanding of this new mode of serine protease inhibition and could provide a basis for the design of novel fXa inhibitors. Analyses of homo- and heteronuclear two-dimensional NMR spectra (total correlation spectroscopy, nuclear Overhauser effect spectroscopy [NOESY], constant time heteronuclear single quantum correlation spectroscopy [CT-HSQC], and HSQC-NOESY; 600 MHz; 1.5 mM TAP; pH 2.5) of unlabeled, 13C-labeled, and 15N-labeled TAP provided nearly complete 1H sequence-specific resonance assignments. Secondary structural elements were identified by characteristic NOE patterns and D2O amide proton-exchange experiments. A three-dimensional structure of TAP was generated from 412 NOESY-derived distance and 47 dihedral angle constraints. The structural elements of TAP are similar in some respects to those of the Kunitz serine protease inhibitor family, with which TAP shares weak sequence homology. This structure, coupled with previous kinetic and biochemical information, confirms previous suggestions that TAP has a unique mode of binding to fXa.



	Figure 1. Fig. 1. Ca region f a'3C-constanttimeHSQCcorrelationexperi- ment.Peaksare labeled by the single-letter codeandsequencenumber, withthe Ca peaksdistinguishedfrom C@ andC6peaks by an enclos- ing box.Theconstanttimeperiod was 17 ms,withatotal of 189 incre- ments,eachasum of 64 transients.Processing was performedwith TRIADsoftware(Tripos Associates). Theintensityofthe first point for achID was halved, followed by apodization with asquared sine wave, shifted by 80". Each FID was zero-filled to 2,048points,Fouriertrans- formed,phased,andthen bascline correted with apolynomialfit.Pairs ofinterferogramsfrom 512 incrementswereshuffled to separaereal ndmaginaryparts.Thesecondtransform was perforedftersimi- lar halvingofthefirstpointandapodization,followed by zerofilling o 1,024 oints.Thespectrum was referenced to theTSP peak t 0 ppm forbothcarbonandproton dimensions; two wl sweepwidths were added to te ormer to obtainthewaleforthisregion.		Figure 9. Fig. 9. Superposition of thebakboneatoms of arefinedTAPstructure(cyan)andBPTI (yellow) (Marquartet al., 1983)alignedusing backboneatoms (N, Ca, , 0) of residues22- 28,32-38, and51-60 of APwithbackbone atoms of residues 18-24,29-35, and47-56 of PTI,respectively.Selectedresiduenumbers of TAP (1-4,30,42,60) andBPTI Ii 14,15, 8, 39, 58)are shown to provideanorienta- tion. Cystinesidechainsaredepicted for each olecule.Solvent-accessiblesurfaces(probe adius 1.4 A) areshown as dotsurfacesforthe riticalbindingresidues of TAP (1-4,42) and BPTI (11, 13-19,34,36-39). Theorientation isidentical to Figures6and 7.