PDBsum entry 1rtf

Serine protease

PDB id

1rtf

Contents

			Protein chain

					244 a.a. *

			Ligands

					THR-CYS-GLY-LEU- ARG-GLN-TYR-SER


					PO4


					BEN

			Waters ×168

* Residue conservation analysis

References listed in PDB file

Key reference

Title

The 2.3 a crystal structure of the catalytic domain of recombinant two-Chain human tissue-Type plasminogen activator.

Authors

D.Lamba, M.Bauer, R.Huber, S.Fischer, R.Rudolph, U.Kohnert, W.Bode.

Ref.

J Mol Biol, 1996, 258, 117-135. [DOI no: 10.1006/jmbi.1996.0238]

PubMed id

8613982

Abstract

Tissue-type plasminogen activator (t-PA), a multidomainal serine proteinase of the trypsin-family, catalyses the rate-limiting step in fibrinolysis, the activation of plasminogen to the fibrin-degrading proteinase plasmin. Trigonal crystals have been obtained of the recombinant catalytic domain of human-two-chain t-PA, consisting of a 17 residue A chain and the 252 residue B chain. Its X-ray crystal structure has been solved applying Patterson and isomorphous replacement methods, and has been crystallographically refined to an R-value of 0.184 at 2.3 A resolution. The chain fold, active-site geometry and Ile276-Asp477 salt bridge are similar to that observed for trypsin. A few surface-located insertion loops differ significantly, however. The disulfide bridge Cys315-Cys384, practically unique to the plasminogen activators, is incorporated without drastic conformational changes as the insertion loop preceding Cys384 makes a bulge on the molecular surface. The unique basic insertion loop Lys296-Arg304 flanking the primed subsites, which has been shown to be of importance for PAI-1 binding and for fibrin specificity, is partially disordered; it can therefore freely adapt to proteins docking to the active site. The S1 pocket of t-PA is almost identical to that of trypsin, whereas the S2 site is considerably reduced in size by the imposing Tyr368 side-chain, in agreement with the measured preference for P1 Arg and P2 Gly residues. The neighbouring S3-S4 hydrophobic groove is mainly hydrophobic in nature. The structure of the proteinase domain of two-chain t-PA suggests that the formation of a salt bridge between Lys429 and Asp477 may contribute to the unusually high catalytic activity of single-chain t-PA, thus stabilizing the catalytically active conformation without unmasking the Ile276 amino terminus. Modeling studies show that the covalently bound kringle 2 domain in full-length t-PA could interact with an extended hydrophobic groove in the catalytic domain; in such a docking geometry its "lysine binding site" and the "fibrin binding patch" of the catalytic domain are in close proximity.



	Figure 3. Figure 3. Stereo section of the final electron density map (blue) around the bound benzamidine molecule (center), superimposed on the t-PA model. Standard view as in Figures 1 and 2. Of the protein structure, only the entrance frame Trpc215 to Cysc220 (around the benzamidine), the catalytic triad Serc195, Hisc57 and Aspc102 (to the east), and Lysc143 and Tyrc151 (to the south) are displayed; the spherical density east of the benzamidine molecule, which partially hides the active Ser195, represents the bound phosphate ion. Contouring is at 1.0s. Figure made with O (Jones et al., 1991).		Figure 5. Figure 5. Stereo plot of the hypothetical docking complex of the catalytic domain (white connections) and kringle 2 (yellow connections; De Vos et al., 1992) in the covalent two-domain t-PA variant. The domains are superimposed with a blue (catalytic domain) and a green Connolly surface (kringle 2 domain). This view is approximately rotated 135° from the standard orientation around a horizontal axis, so that the active site is now pointing to the east/back. Charged side-chains of catalytic domain residues presumably involved in fibrin binding, and kringle 2 residues forming the lysine binding site are labeled (chymotrypsinogen and kringle nomenclature). The plot was made with MAIN (Turk, 1992).

PROCHECK

	Headers