Chymotryptic specificity determinants in the 1.0 A structure of the zinc-inhibited human tissue kallikrein 7

Abstract

hK7 or human stratum corneum chymotryptic enzyme belongs to the human tissue kallikrein (hKs) serine proteinase family and is strongly expressed in the upper layers of the epidermis. It participates in skin desquamation but is also implicated in diverse skin diseases and is a potential biomarker of ovarian cancer. We have solved x-ray structures of recombinant active hK7 at medium and atomic resolution in the presence of the inhibitors succinyl-Ala-Ala-Pro-Phe-chloromethyl ketone and Ala-Ala-Phe-chloromethyl ketone. The most distinguishing features of hK7 are the short 70-80 loop and the unique S1 pocket, which prefers P1 Tyr residues, as shown by kinetic data. Similar to several other kallikreins, the enzyme activity is inhibited by Zn(2+) and Cu(2+) at low micromolar concentrations. Biochemical analyses of the mutants H99A and H41F confirm that only the metal-binding site at His(99) close to the catalytic triad accounts for the noncompetitive Zn(2+) inhibition type. Additionally, hK7 exhibits large positively charged surface patches, representing putative exosites for prime side substrate recognition.

Fig. 1.

Inhibition kinetics of hK7. (A) Inhibition curve of hK7 measured with the fluorogenic substrate Suc-AAPF-AMC and zinc and copper concentrations from 0.2 to 500 μM. The fractional activity is plotted against the logarithmic values of the metal ion concentration. (B) The Lineweaver–Burk plot for varying Suc-LLY-AMC concentrations and 0, 5, 10, and 15 μM Zn²⁺ demonstrates the noncompetitive inhibition type of hK7. (C) Activity of the hK7 mutants H41F (■) and H99A (open hexagons) against Suc-LLY-AMC with varying Zn²⁺ concentrations. The H99A mutation abolished the inhibitory effect of Zn²⁺ completely, whereas the H41F mutation caused no change with respect to the wild type.

Fig. 2.

Tertiary and primary structure of hK7. (A) Overall structure of hK7_E monomer with the AAF-CMK inhibitor in a stereo ribbon representation. Side chains of the catalytic triad (His⁵⁷, Asp¹⁰², Ser¹⁹⁵), the specificity-determining Asn¹⁸⁹ in the S1 pocket, the oxyanion hole forming Gly¹⁹³, and the double covalently bound inhibitor are depicted as stick models. Important loops are labeled and highlighted in green. (B) Electrostatic surface representation for hK7_I in a hypothetical complex with the modeled peptide Glu-Ala-Leu-Tyr-Leu-Val, occupying the specificity sites S4 to S2′. (C) Sequence alignment of hK7 with the tissue kallikreins hK6, hK1, hK5, and bovine chymotrypsin as a reference for numbering. Residues involved in metal binding are indicated by magenta background, whereas loops are surrounded by boxes.

Fig. 3.

Comparison of the hK7 active site at medium and atomic resolution. (A) Active site of hK7_I with the Suc-AAPF-CMK inhibitor as stick model in stereo at 2.0 Å resolution with a 2F_o − F_c map contoured at 1.0σ. The inhibitor is covalently bound to the Nε of His⁵⁷ and the Oγ of Ser¹⁹⁵ and corresponds to substrate residues P4–P1. (B) The same view of the inhibitor at 1.0 Å resolution represented as a ball-and-stick model with a 2F_o − F_c map contoured at 1.0σ as transparent surface and contoured at 3.5σ as orange grid. Two alternative conformations with approximately equal occupancy are seen for the inhibitor Ala² side chain and the carboxylate of the succinyl group. In the Trp²¹⁵ side chain, density for hydrogen atoms (white balls) is observed.

Fig. 4.

Stick model of the modeled substrate Glu-Ala-Leu-Tyr-Leu-Val, the catalytic triad and the backbone of Gly¹⁹³, Ser¹⁹⁵, and Gly²¹⁶ of hK7_I in stereo, including hydrogen bonds as dotted lines. The S1 pocket is depicted as transparent green surface according to volume calculations with VOIDOO (41). The specificity for P1 Tyr is most likely conferred by Asn¹⁸⁹ via hydrogen bonds from the carboxamide side chain to an interconnecting water molecule and to the Tyr OH group, respectively.

Fig. 5.

The copper ions bound at His⁹⁹ and His⁴¹ displayed as blue spheres surrounded by electron density of the anomalous Fourier map in red (contour 5σ) in stereo. His⁵⁷ has the capacity for liganding Cu1 and Cu2 by a side chain rotation (His57*), requiring shifts of the ions, whereas the mutant H99A proves that only the His⁹⁹ site is the structural basis for the Zn²⁺ and Cu²⁺ inhibition of hK7.