Chymotryptic specificity determinants in the 1.0 A structure of the zinc-inhibited human tissue kallikrein 7 (original) (raw)
Related papers
Structural Basis of the Zinc Inhibition of Human Tissue Kallikrein 5
Journal of Molecular Biology, 2007
Human kallikrein 5 (hK5) is a member of the tissue kallikrein family of serine peptidases. It has trypsin-like substrate specificity, is inhibited by metal ions, and is abundantly expressed in human skin, where it is believed to play a central role in desquamation. To further understand the interaction of hK5 with substrates and metal ions, active recombinant hK5 was crystallized in complex with the tripeptidyl aldehyde inhibitor leupeptin, and structures at 2.3 Å resolution were obtained with and without Zn 2+ . While the overall structure and the specificity of S1 pocket for basic sidechains were similar to that of hK4, a closely related family member, both differed in their interaction with Zn 2+ . Unlike hK4, the 75-loop of hK5 is not structured to bind a Zn 2+ . Instead, Zn 2+ binds adjacent to the active site, becoming coordinated by the imidazole rings of His99 and His96 not present in hK4. This zinc binding is accompanied by a large shift in the backbone conformation of the 99-loop and by large movements of both His side-chains. Modeling studies show that in the absence of bound leupeptin, Zn 2+ is likely further coordinated by the imidazolyl side-chain of the catalytic His57 which can, similar to equivalent His57 imidazole groups in the related rat kallikrein proteinase tonin and in an engineered metalbinding rat trypsin, rotate out of its triad position to provide the third coordination site of the bound Zn 2+ , rendering Zn 2+ -bound hK5 inactive. In solution, this mode of binding likely occurs in the presence of free and substrate saturated hK5, as kinetic analyses of Zn 2+ inhibition indicate a non-competitive mechanism. Supporting the His57 re-orientation, Zn 2+ does not fully inhibit hK5 hydrolysis of tripeptidyl substrates containing a P2-His residue. The P2 and His57 imidazole groups would lie next to each other in the enzyme-substrate complex, indicating that incomplete inhibition is due to competition between both imidazole groups for Zn 2+ . The His96-99-57 triad is thus suggested to be responsible for the Zn 2+mediated inhibition of hK5 catalysis.
Journal of Investigative Dermatology
Keratins 1 (K1) and 10 (K10) are the primary keratins expressed in differentiated epidermis. Mutations in K1/K10 are associated with human skin diseases. We determined the crystal structure of the complex between the distal (2B) helices of K1 and K10 to better understand how human keratin structure correlates with function. The 3.3 Å resolution structure confirms many features inferred by previous biochemical analyses, but adds unexpected insights. It demonstrates a parallel, coiled-coil heterodimer with a predominantly hydrophobic intermolecular interface; this heterodimer formed a higher order complex with a second K1-K10-2B heterodimer via a Cys401 K10 disulfide link, although the bond angle is unanticipated. The molecular surface analysis of K1-K10-2B identified several pockets, one adjacent to the disulfide linkage and conserved in K5-K14. The solvent accessible surface area of the K1-K10 structure is 20e25% hydrophobic. The 2B region contains mixed acidic and basic patches proximally (N-terminal), whereas it is largely acidic distally (C-terminal). Mapping of conserved and nonconserved residues between K1-K10 and K5-K14 onto the structure demonstrated the majority of unique residues align along the outer helical ridge. Finally, the structure permitted a fresh analysis of the deleterious effects caused by K1/K10 missense mutations found in patients with phenotypic skin disease.
Specificity Profiling of Seven Human Tissue Kallikreins Reveals Individual Subsite Preferences
Journal of Biological Chemistry, 2006
Human tissue kallikreins (hKs) form a family of 15 closely related (chymo)trypsin-like serine proteinases. These tissue kallikreins are expressed in a wide range of tissues including the central nervous system, the salivary gland, and endocrine-regulated tissues, such as prostate, breast, or testis, and may have diverse physiological functions. For several tissue kallikreins, a clear correlation has been established between expression and different types of cancer. For example, the prostate-specific antigen (PSA or hK3) serves as tumor marker and is used to monitor therapy response. Using a novel strategy, we have cloned, expressed in Escherichia coli or in insect cells, refolded, activated, and purified the seven human tissue kallikreins hK3/PSA, hK4, hK5, hK6, hK7, hK10, and hK11. Moreover, we have determined their extended substrate specificity for the nonprime side using a positional scanning combinatorial library of tetrapeptide substrates. hK3/PSA and hK7 exhibited a chymotrypsin-like specificity preferring large hydrophobic or polar residues at the P1 position. In contrast, hK4, hK5, and less stringent hK6 displayed a trypsin-like specificity with strong preference for P1-Arg, whereas hK10 and hK11 showed an ambivalent specificity, accepting both basic and large aliphatic P1 residues. The extended substrate specificity profiles are in good agreement with known substrate cleavage sites but also in accord with experimentally solved (hK4, hK6, and hK7) or modeled structures. The specificity profiles may lead to a better understanding of human tissue kallikrein functions and assist in identifying their physiological protein substrates as well as in designing more selective inhibitors. The abbreviations used are: PSA, prostate-specific antigen; hK, human tissue kallikrein; EK, enterokinase; AMC, 7-amino-4-methylcoumarine; uPA, urokinase-type plasminogen activator; PS-SCL, positional scanning with a synthetic combinatorial peptide library; BPTI, bovine pancreatic trypsin inhibitor.
European Journal of Medicinal Chemistry, 2013
Netherton syndrome is caused by loss-of-function mutations in SPINK5 encoding the Kazal-type inhibitor LEKTI-1 leading to dysregulation of proteolytic cascades involving several kallikreins. We used both structure-based and ligand-based virtual screening computations to identify commercially available noncovalent inhibitors of human kallikrein 5 (hK5), a serine protease (trypsin-like) that plays a central role in the initiation of the molecular cascades leading to the Netherton syndrome phenotype. The efficacy and mechanism of inhibition of the identified new families of organic compounds were analyzed not only for hK5 but also on other proteases implicated in the cascades (hK7, hK14 and matriptase). These inhibitors are nontoxic on healthy human keratinocytes and are structurally different from traditional serine protease inhibitors validating their potential utility as initial hits to control proteolytic disorders observed in dermatological pathologies such as Netherton syndrome.
Biochemical Journal, 2003
We have demonstrated that the S 1 and S 2 subsites of human tissue kallikrein (hK1) play determinant roles in the recognition and hydrolysis of substrates. The presence of serine at position P 1 and arginine at P 2 resulted in the best substrate, Abz-Ala-Ile-Lys-Phe-Phe-Ser-Arg-Gln-EDDnp, which was derived from the kallistatin reactive-centre loop sequence and quencher groups o-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp). Serine and arginine are also the residues at positions P 1 and P 2 in human kininogen, from which hK1 releases Lys-bradykinin. Several peptide analogues of Abz-Ala-Ile-Lys-Phe-Phe-Ser-Arg-Gln-EDDnp, in which the Ser and Arg residues were substituted with various other amino acids, were synthesized and tested as substrates. Most of them were hydrolysed slowly, although they showed significant binding to hK1, as demonstrated by their competitive inhibition constants (K i ). Using this information, six peptides were designed, synthesized and assayed as inhibitors of hK1. Abz-Lys-Phe-Phe-Pro-Arg-Gln-EDDnp, Abz-Lys-Phe-Arg-Pro-Arg-Gln-EDDnp and acetyl-Lys-Phe-Phe-Pro-Leu-Glu-NH 2 inhibited hK1 in the range 20-30 nM (letters in italics denote the D-form of the amino acid). The peptide acetyl-Lys-Phe-Phe-Pro-Leu-Glu-NH 2 was a weak inhibitor for other serine proteases, as indicated by the higher K i values compared with hK1, but this peptide was a potent inhibitor of human plasma kallikrein, which has a K i value of 8 nM. This result was surprising, since this enzyme is known to be a restricted arginyl-hydrolase. In conclusion, acetyl-Lys-Phe-Phe-Pro-Leu-Glu-NH 2 can be used as a leader compound to design specific inhibitors for hK1, plasma kallikrein, or for both at same time, if the inhibition of kinin release is the main goal.
European Journal of Biochemistry, 1999
Human glandular kallikrein 2 (hK2) is a serine protease expressed by the prostate gland with 80% identity in primary structure to prostate-specific antigen (PSA). Recently, hK2 was shown to activate the zymogen form of PSA (proPSA) in vitro and is likely to be the physiological activator of PSA in the prostate. hK2 is also able to activate urokinase and effectively cleave fibronectin. We studied the substrate specificity of hK2 and regulation of its activity by zinc and extracellular protease inhibitors present in the prostate and seminal plasma. The enzymatic activity and substrate specificity was studied by determining hK2 cleavage sites in the major gel proteins in semen, semenogelin I and II, and by measuring hydrolysis of various tripeptide aminomethylcoumarin substrates. HK2 cleaves substrates C-terminal of single or double arginines. Basic amino acids were also occasionally found at several other positions N-terminal of the cleavage site. Therefore, the substrate specificity of hK2 fits in well with that of a processor of protein precursors. Possible regulation mechanisms were studied by testing the ability of Zn 2+ and different protease inhibitors to inhibit hK2 by kinetic measurements. Inhibitory constants were determined for the most effective inhibitors PCI and Zn 2+ . The high affinity of PCI for hK2 (k ass = 2.0 Â 10 5 m 21´s21 ) and the high concentrations of PCI (4 mm) and hK2 (0.2 mm) in seminal plasma make hK2 a very likely physiological target protease for PCI. hK2 is inhibited by Zn 2+ at micromolar concentrations well below the 9 mm zinc concentration found in the prostate. The enzymatic activity of hK2 is likely to be reversibly regulated by Zn 2+ in prostatic fluid. This regulation may be impaired in CAP and advanced metastatic cancer resulting in lack of control of the hK2 activity and a need for other means of control.
Biological Chemistry, 2005
LEKTI is a 120-kDa protein that plays an important role in skin development, as mutations affecting LEKTI synthesis underlie Netherton syndrome, an inherited skin disorder producing severe scaling. Its primary sequence indicates that the protein consists of 15 domains, all resembling a Kazal-type serine protease inhibitor. LEKTI and two serine proteases belonging to the human tissue kallikrein (hK) family (hK5 and hK7) are expressed in the granular layer of skin. In this study, we characterize the interaction of two recombinant LEKTI fragments containing three or four intact Kazal domains (domains 6–8 and 9–12) with recombinant rhK5, a trypsin-like protease, and recombinant rhK7, a chymotrypsin-like protease. Both fragments inhibited rhK5 similarly in binding and kinetic studies performed at pH 8.0, as well as pH 5.0, the pH of the stratum corneum where both LEKTI and proteases may function. Inhibition equilibrium constants (Ki) measured either directly in concentration-dependent st...