Abstract 5344: Understanding the structural requirements of C1 domains for phorbol ester binding, analysis of the atypical C1 domain of STAC2
Authors: Abramovitz Adelle , Kedei Noemi , Blumberg Peter M 
Published: July 2017

Abstract
C1 domains are the recognition motif for the second messenger diacylglycerol and for the phorbol esters. They play a critical role in the regulation of multiple families of signaling proteins such as protein kinase C, RasGRP, and the chimaerins. Atypical C1 domains preserve substantial homology with the diacylglycerol / phorbol ester responsive C1 domains but fail to bind these ligands. A recent advance in the field has been the recognition that the atypical C1 domains represent a continuum of structures ranging from those with measurable but weak phorbol ester binding affinity through those that preserve the phorbol ester binding cleft but have residues interfering with the insertion of the C1 domain into membranes and finally those that lack appropriate binding cleft geometry. STAC2 (SH3 and cysteine-rich domain 2) protein attracted our interest because, while its C1 domain has been classified as atypical, it had actually not been tested for phorbol ester binding activity and possesses marked homology to C1 domains that bind phorbol esters. The strategy for analysis has been to probe the influence of individual residues in the STAC2 C1 domain that are either not present or are unusual in the numerous phorbol ester responsive C1 domains. Reciprocal approaches have been to start either with the STAC2 C1 domain, mutate the individual residues, and look for gain of phorbol ester binding activity or to start with the PKC delta C1b domain and look for residues that cause reduction in binding affinity. The former approach is efficient if a single residue is responsible for the lack of binding activity; the latter approach is preferable if multiple residues are responsible. First, we confirmed that indeed the C1 domain of STAC2 is atypical and fails to bind phorbol ester. Second, we identified the residues in positions 11, 13, 24 25, 41 and 45 of the C1 domain of STAC2 as potential candidates for interfering with binding activity. However, replacement of none of these residues with the corresponding residues from PKC delta C1b sufficed to generate phorbol ester binding activity. One the other hand, introducing mutations into the C1b domain of PCK delta identified L24N, that caused a significant (~20-fold) decrease in the binding affinity. The probable basis for the negative effect of L24N on binding is that Leucine 24, together with Phenylalanine 13, Leucine 20 and Tryptophan 22, are all exposed at the top of the binding cleft and insert into the lipid membrane during ligand binding. The replacement of a hydrophobic residue at position 24 with a hydrophilic residue should interfere with this binding. STAC2 may thus fall into the category of proteins with atypical C1 domains that could be targeted with phorbol derivatives that do not stabilize membrane insertion. Our findings strengthen our insight into the structural basis and understanding of the interplay between the C1 domain, ligand and the membrane as a ternary complex.
Citation Format: Adelle Abramovitz, Noemi Kedei, Peter M Blumberg. Understanding the structural requirements of C1 domains for phorbol ester binding, analysis of the atypical C1 domain of STAC2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5344. doi:10.1158/1538-7445.AM2017-5344