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Abstract
A key challenge in studying protein/protein interactions is to accurately identify contact surfaces, i.e. regions of two proteins that are in direct physical contact. Aside from x-ray crystallography and NMR spectroscopy few methods are available that address this problem. Although x-ray crystallography often provides detailed information about contact surfaces, it is limited to situations when a co-crystal of proteins is available. NMR circumvents this requirement but is limited to small protein complexes. Other methods, for instance protection from proteolysis, are less direct and therefore less informative. Here we describe a new method that identifies candidate contact surfaces in protein complexes. The complexes are first stabilized by cross-linking. They are then digested with a protease, and the cross-linked fragments are analyzed by mass spectrometry. We applied this method, referred to as COSUMAS (contact surfaces by mass spectrometry), to two proteins, retinal guanylyl cyclase 1 (RetGC1) and guanylyl cyclase-activating protein-1 (GCAP-1), that regulate cGMP synthesis in photoreceptors. Two regions in GCAP-1 and three in RetGC1 were identified as possible contact sites. The two regions of RetGC1 that are in the vicinities of Cys(741) and Cys(780) map to a kinase homology domain in RetGC1. Their identities as contact sites were independently evaluated by peptide inhibition analysis. Peptides with sequences from these regions block GCAP-1-mediated regulation of guanylyl cyclase at both high and low Ca2+ concentrations. The two regions of GCAP-1 cross-linked to these peptides were in the vicinities of Cys(17) and Cys(105) of GCAP-1. Peptides with sequences derived from these regions inhibit guanylyl cyclase activity directly. These results support a model in which GCAP-1 binds constitutively to RetGC1 and regulates cyclase activity by structural changes caused by the binding or dissociation of Ca2+.
Highlights
RetGC11 and RetGC2, are membrane guanylyl cyclases that synthesize cGMP within the outer segments of rod and cone
Crosslinked proteins were separated by SDS-polyacrylamide gel electrophoresis, and the ability of bMH to stabilize RetGC11⁄7GCAP-1 complexes was assessed by Western blotting with anti-retinal guanylyl cyclase 1 (RetGC1) and -guanylyl cyclase-activating protein-1 (GCAP-1) antibodies
In this study we provide evidence for multiple contact surfaces involved in RetGC1/GCAP-1 interaction (Fig. 9)
Summary
RetGC11 and RetGC2, are membrane guanylyl cyclases that synthesize cGMP within the outer segments of rod and cone. Lange and coworkers [17] identified two different regions in RetGC1, 492LHIQMVSGPNKIILTLDDIT511 and 553LYEGDWVWLKKFPGDRHIAI571, as sites of contact with GCAP-1 These sequences lie within the intracellular region between the membrane and the kinase homology domain of RetGC1. Both studies relied on peptide binding and inhibition analyses to identify contact surfaces. Regions of GCAPs that are important for regulation of RetGCs have been studied primarily by mutagenesis of GCAP-1 and GCAP-2 (11, 12, 18 –21) None of those studies provided direct evidence for sites of interaction with RetGCs. Additional information about interactions between RetGCs and GCAPs comes from proteolysis of RetGC chimeras [22] and stabilization of the kinase homology domain by GCAPs [23]. Stabilization and protection from proteolysis occurred independently of Ca2ϩ suggesting that GCAPs bind constitutively to RetGCs
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