Crystal Structure of a Prostate Kallikrein Isolated from Stallion Seminal Plasma: A Homologue of Human PSA

Abstract

Prostate-specific kallikrein, a member of the gene family of serine proteases, was initially discovered in semen and is the most useful serum marker for prostate cancer diagnosis and prognosis. We report the crystal structure at 1.42 Å resolution of horse prostate kallikrein (HPK). This is the first structure of a serine protease purified from seminal plasma. HPK shares extensive sequence homology with human prostate-specific antigen (PSA), including a predicted chymotrypsin-like specificity, as suggested by the presence of a serine residue at position S1 of the specificity pocket. In contrast to other kallikreins, HPK shows a structurally distinct specificity pocket. Its entrance is blocked by the kallikrein loop, suggesting a possible protective or substrate-selective role for this loop. The HPK structure seems to be in an inactivated state and further processing might be required to allow the binding of substrate molecules. Crystal soaking experiments revealed a binding site for Zn²⁺ and Hg²⁺, two known PSA inhibitors.

Introduction

Kallikreins are members of the mammalian serine protease gene family and participate in a significant number of carefully controlled physiological processes such as blood clotting, fibrinolysis, fertilization and hormone production. Glandular kallikrein (prostate-specific antigen, PSA) is an androgen-regulated secretory product of the prostate epithelium. PSA participates in the lysis of the seminal coagulum formed upon ejaculation when prostatic and seminal vesicle fluids are mixed.¹ This involves the cleavage of semenogelins I and II proteins at tyrosine and leucine residues. Accumulating evidence indicates that members of the kallikrein gene family are related to the pathogenesis of human diseases, depending on the tissue of their primary expression, including inflammation, hypertension, renal nephritis and diabetic renal disease, pathological keratinization and psoriasis, epilepsy, the development of Alzheimer's disease, the pathophysiology of the thyroid, and malignancy.2., 3. In particular, the prostate-specific antigen is currently the best tumor marker for prostate cancer,⁴ the most frequently diagnosed cancer in men. It can be considered indicative of benign conditions, such as bacterial prostatitis, urinary retention and benign prostatic hyperplasia.5., 6. It has been shown that the concentration of serum PSA is proportional to tumor volume and correlates positively with the clinical stage of the disease.⁵

In prostate tissue and in seminal plasma, PSA exists predominantly in a free form,⁶ although its catalytic activity is inhibited by Zn²⁺, spermine, and spermidine, each a major component of seminal and prostatic fluid, with Zn²⁺ being a non-competitive inhibitor, while spermine and spermidine are competitive inhibitors. In serum, PSA has been found mainly complexed to protease inhibitors such as α₂-macroglobulin, α₁-antichymotrypsin and α₁-proteinase inhibitor,7., 8., 9. and the proteolytic activity of PSA appears to be restricted to the microenvironment surrounding prostate cancer cells¹⁰ where the continuous release of active PSA into interstitial fluid can build to appreciable steady-state levels. However, the physiological function of PSA and the connection of its catalytic activity to the pathogenesis and development of prostate cancer are likely to be complex and are not well understood. A number of pieces of evidence indicate that PSA may be deleterious to breast, prostate, and other tissues or that PSA is a beneficial molecule with tumor-suppressor activity. PSA has been reported to cleave insulin-like growth factor-binding protein-3,11., 12. liberating IGF-1, which is a known mitogen of many cell types and a risk factor for prostate and breast cancer development.¹³ PSA activates single-chain urokinase-type plasminogen activator,¹⁴ which is linked with prostate cancer invasion and metastasis.¹⁵ Other substrates include fibronectin and laminin (linked to proliferation of prostatic stromal and epithelial cells, tumor spread, invasion, and metastasis),¹⁶ and parathyroid-hormone-related protein,¹⁷ further supporting a role for PSA as a promoter of cancer cell proliferation.¹⁸ On the other hand, other publications indicate that PSA may have beneficial properties as a negative regulator of cell growth, an anticarcinogenic/antiangiogenic molecule, or as an inducer of apoptosis.¹⁹ These results suggest that both targeted upregulation and downregulation of PSA activity in vivo could have relevant therapeutic applications.

At the sequence level, a distinct feature of PSA is the presence of serine 189 at the bottom of the substrate-specificity pocket, which is a major determinant of its chymotrypsin-like preference for cleaving substrates on the carboxyl side of hydrophobic residues.1., 10., 20., 21. However, a detailed knowledge of its 3D structure is central to understanding PSA activity and its regulation in order for optimizing attempts to use PSA-specific pro-drugs for the treatment of advanced prostate cancer.22., 23. In the absence of a crystal structure, homology models based on structural alignments with other serine proteases of known X-ray crystallographic structures (rat submandibular gland tonin, 1TON;²⁴ pancreatic kallikrein, 2PKA;²⁵ chymotrypsin, 5CHA;²⁶ and trypsin, 1TLD²⁷) have been proposed (PDB accession codes 1PFA and 2PSA).18., 28., 29. PSA shares high sequence similarity and the structural framework of serine proteases, which consists mainly of two six-stranded antiparallel β-barrels with the catalytic triad (His57, Asp102, and Ser195), the oxyanion hole, and main-chain substrate-binding residues located in a cleft between the two barrels. However, a unique feature of PSA, like the kallikrein loop inserted in the region of residue 95, was modeled using molecular dynamics and electrostatic calculations. As noted by the authors,²⁹ non-homologous loop building presents a major problem in homology modeling, and the uncertainty of the model is greatest in these regions. Now, we report the crystal structure of horse prostate kallikrein (HPK), the first structure of a serine protease purified from mammalian seminal plasma. The significant sequence homology to human PSA and the prostatic origin of HPK, suggest that this protein represents the equine counterpart of human PSA. In agreement with the proposed models, the residue at the bottom of the substrate-specificity/recognition pocket of HPK is serine (Ser189) (Figure 1), conferring to HPK a predicted chymotrypsin-like activity. Nevertheless, there are significant structural differences between the specificity pockets of HPK and other serine proteases, which will be discussed. HPK is the first crystal structure of a PSA and its distinct structural features among kallikreins make it a much better model for human PSA than any other kallikrein structure reported to date. Structural comparisons between HPK, PSA models, and known structures of the serine protease family are presented, and the structures of two HPK with bound PSA inhibitors, Zn²⁺ (Zn-HPK) and Hg²⁺ (Hg-HPK), are described.