BACKGROUND: Human Prostate Secretory Protein of 94 amino acids (PSP94) has been shown to bind human CRISP-3 (cysteine-rich secretory protein 3) with very high affinity. CRISP-3 belongs to the CRISP family of proteins having a PR-1 (pathogenesis related protein 1) domain at its N-terminal and ion channel regulatory (ICR) domain at its C-terminal connected by a hinge region. Functional significance of this complex is not yet known. METHODS: In order to identify the residues and/or regions involved in PSP94-CRISP-3 interaction, site-directed mutagenesis was employed. Effect of the mutations on the interaction was studied by co-immunoprecipitation (Co-IP). RESULTS: For PSP94, amino acids Y(3), F(4), P(56) and the C-terminal β-strand were found to be crucial for interacting with CRISP-3. A disulfide bond between the two domains of PSP94 (C(37)A-C(73)A) was also important for this interaction. In case of CRISP-3, the N-terminal domain alone could not maintain a strong interaction with PSP94 but it required presence of the hinge region and not the C-terminal domain. Apart from CRISP-3, CRISP-2 was also found to interact with human PSP94. Based on our findings the most likely model of PSP94-CRISP-3 complex has been proposed. CONCLUSION: The terminal β-strands of PSP94 contact the first α-helix and the hinge region of CRISP-3. GENERAL SIGNIFICANCE: Involvement of the hinge region of CRISPs in interaction with PSP94 may affect the domain movement of CRISPs essential for the ion-channel regulatory activity resulting in inhibition of this activity.
BACKGROUND:HumanProstate Secretory Protein of 94 amino acids (PSP94) has been shown to bind humanCRISP-3 (cysteine-rich secretory protein 3) with very high affinity. CRISP-3 belongs to the CRISP family of proteins having a PR-1 (pathogenesis related protein 1) domain at its N-terminal and ion channel regulatory (ICR) domain at its C-terminal connected by a hinge region. Functional significance of this complex is not yet known. METHODS: In order to identify the residues and/or regions involved in PSP94-CRISP-3 interaction, site-directed mutagenesis was employed. Effect of the mutations on the interaction was studied by co-immunoprecipitation (Co-IP). RESULTS: For PSP94, amino acids Y(3), F(4), P(56) and the C-terminal β-strand were found to be crucial for interacting with CRISP-3. A disulfide bond between the two domains of PSP94 (C(37)A-C(73)A) was also important for this interaction. In case of CRISP-3, the N-terminal domain alone could not maintain a strong interaction with PSP94 but it required presence of the hinge region and not the C-terminal domain. Apart from CRISP-3, CRISP-2 was also found to interact with humanPSP94. Based on our findings the most likely model of PSP94-CRISP-3 complex has been proposed. CONCLUSION: The terminal β-strands of PSP94 contact the first α-helix and the hinge region of CRISP-3. GENERAL SIGNIFICANCE: Involvement of the hinge region of CRISPs in interaction with PSP94 may affect the domain movement of CRISPs essential for the ion-channel regulatory activity resulting in inhibition of this activity.