PDBsum entry 1ets

Hydrolase/hydrolase inhibitor

PDB id: 1ets

Contents

Protein chains

36 a.a. *

259 a.a. *

Ligands

MID

Waters ×684

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Refined 2.3 a X-Ray crystal structure of bovine thrombin complexes formed with the benzamidine and arginine-Based thrombin inhibitors napap, 4-Tapap and mqpa. A starting point for improving antithrombotics.

Authors

H.Brandstetter, D.Turk, H.W.Hoeffken, D.Grosse, J.Stürzebecher, P.D.Martin, B.F.Edwards, W.Bode.

Ref.

J Mol Biol, 1992, 226, 1085-1099. [DOI no: 10.1016/0022-2836(92)91054-S]

PubMed id

1518046

Abstract

Well-diffracting crystals of bovine epsilon-thrombin in complex with several "non-peptidic" benzamidine and arginine-based thrombin inhibitors have been obtained by co-crystallization. The 2.3 A crystal structures of three complexes formed either with NAPAP, 4-TAPAP, or MQPA, were solved by Patterson search methods and refined to crystallographic R-values of 0.167 to 0.178. The active-site environment of thrombin is only slightly affected by binding of the different inhibitors; in particular, the exposed "60-insertion loop" essentially maintains its typical projecting structure. The D-stereoisomer of NAPAP and the L-stereoisomer of MQPA bind to thrombin with very similar conformations, as previously inferred from their binding to bovine trypsin; the arginine side-chain of the latter inserts into the specificity pocket in a "non-canonical" manner. The L-stereoisomer of 4-TAPAP, whose binding geometry towards trypsin was only poorly defined, is bound to the thrombin active-site in a compact conformation. In contrast to NAPAP, the distal p-amidino/guanidino groups of 4-TAPAP and MQPA do not interact with the carboxylate group of Asp189 in the thrombin specificity pocket in a "symmetrical" twin N-twin O manner, but through "lateral" single N-twin O contacts; in contrast to the p-amidino group of 4-TAPAP, however, the guanidyl group of MQPA packs favourably in the pocket due to an elaborate hydrogen bond network, which includes two entrapped water molecules. These thrombin structures confirm previous conclusions of the important role of the intermolecular hydrogen bonds formed with Gly216, and of the good sterical fit of the terminal bulky hydrophobic inhibitor groups with the hydrophobic aryl binding site and the S2-cavity, respectively, for tight thrombin active site binding of these non-peptidic inhibitors. These accurate crystal structures are presumed to be excellent starting points for the design and the elaboration of improved antithrombotics.

Figure 1.
Figure 1. Chemical ormulas of NAPAP (with residues XasIl. GlypI2, Papal3 and Pipn14): (~cr-(2-napthyl-s~Ilphotl~l-

Figure 4.
Figure 4. ctive site region f the complex formed between NAPAP (thick lines) and bovine thrombin (thin lines) superimposed with the final 2F,,, -Fcslf electron denity. The view is (similar to Figs 2 and 3) towards the thrombin surface; the active site residues are o the ight, the specificity pocket is in the back. Contou surface is at @9a.

The above figures are reprinted by permission from Elsevier: J Mol Biol (1992, 226, 1085-1099) copyright 1992.

Secondary reference #1

Title

Crystallographic determination of thrombin complexes with small synthetic inhibitors as a starting point for the receptor-Based design of antithrombotics.

Authors

M.Bauer, H.Brandstetter, D.Turk, J.Stürzebecher, W.Bode.

Ref.

Semin Thromb Hemost, 1993, 19, 352-360. [DOI no: 10.1055/s-2007-993286]

PubMed id

8140427

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 percentage match of 88%.

Secondary reference #2

Title

X-Ray crystal structures of thrombin in complex with d-Phe-Pro-Arg and with small benzamidine- And arginine-Based "non-Peptidic" inhibitors.

Author

W.Bode.

Ref.

Adv Exp Med Biol, 1993, 340, 15-26.

PubMed id

8154332

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 percentage match of 95%.

Secondary reference #3

Title

The refined 1.9-A X-Ray crystal structure of d-Phe-Pro-Arg chloromethylketone-Inhibited human alpha-Thrombin: structure analysis, Overall structure, Electrostatic properties, Detailed active-Site geometry, And structure-Function relationships.

Authors

W.Bode, D.Turk, A.Karshikov.

Ref.

Protein Sci, 1992, 1, 426-471. [DOI no: 10.1002/pro.5560010402]

PubMed id

1304349

Figure 3.
Fig. 3. Tosyl-m-amidinophenylalanyl-piperidine (thickconnections), NAPAP (mediumconnections),and MQPA (thincon- nections)boundtotheactivesite of humana-thrombindisplayedtogetherwiththeConnollysurface f thrombin(Turk et al., 1991). The naphthyl/toluene/chinolyl groups of theinhibitorsinteractwiththearyl-bindingsiteofthrombin;thesidechains ofthe m- and thep-amidinophenylalanyl residues andofthe arginylresidueenterthespecificitypocketfrom slightly differing sites; the S2 subsiteofthrombin is occupiedtodifferentextentsbythe(partiallysubstituted)piperidinemoieties.The viewis similartothestandard view of Figure .

Figure 30.
Fig. 30. Luzzatiplot f thefinalthrombinmodelafterX-PLOR refinement.

The above figures are reproduced from the cited reference with permission from the Protein Society

Secondary reference #4

Title

Geometry of binding of the n alpha-Tosylated piperidides of m-Amidino-, P-Amidino- And p-Guanidino phenylalanine to thrombin and trypsin. X-Ray crystal structures of their trypsin complexes and modeling of their thrombin complexes.

Authors

D.Turk, J.Stürzebecher, W.Bode.

Ref.

FEBS Lett, 1991, 287, 133-138. [DOI no: 10.1016/0014-5793(91)80033-Y]

PubMed id

1879520

Secondary reference #5

Title

Geometry of binding of the benzamidine- And arginine-Based inhibitors n alpha-(2-Naphthyl-Sulphonyl-Glycyl)-Dl-P-Amidinophenylalanyl-Pipe ridine (napap) and (2r,4r)-4-Methyl-1-[N alpha-(3-Methyl-1,2,3,4-Tetrahydro-8- Quinolinesulphonyl)-L-Arginyl]-2-Piperidine carboxylic acid (mqpa) to human alpha-Thrombin. X-Ray crystallographic determination of the napap-Trypsin complex and modeling of napap-Thrombin and mqpa-Thrombin.

Authors

W.Bode, D.Turk, J.Stürzebecher.

Ref.

Eur J Biochem, 1990, 193, 175-182.

PubMed id

2226434

Secondary reference #6

Title

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.

Authors

W.Bode, I.Mayr, U.Baumann, R.Huber, S.R.Stone, J.Hofsteenge.

Ref.

Embo J, 1989, 8, 3467-3475.

PubMed id

2583108