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Degenerate specificity of PDZ domains from RhoA-specific nucleotide exchange factors PDZRhoGEF and LARG

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Smietana K., Kasztura M., Paduch M., Derewenda U., Derewenda Z., Otlewski J.

2008

vol. 55

issue 2

269-280

PDZ domains are ubiquitous protein-protein interaction modules which bind short, usually carboxyterminal fragments of receptors, other integral or membrane-associated proteins, and occasionally cytosolic proteins. Their role in organizing multiprotein complexes at the cellular membrane is crucial for many signaling pathways, but the rules defining their binding specificity are still poorly understood and do not readily explain the observed diversity of their known binding partners. Two homologous RhoA-specific, multidomain nucleotide exchange factors PDZRhoGEF and LARG contain PDZ domains which show a particularly broad recognition profile, as suggested by the identification of five diverse biological targets. To investigate the molecular roots of this phenomenon, we constructed a phage display library of random carboxyterminal hexapeptides. Peptide variants corresponding to the sequences identified in library selection were synthesized and their affinities for both PDZ domains were measured and compared with those of peptides derived from sequences of natural partners. Based on the analysis of the binding sequences identified for PDZRhoGEF, we propose a sequence for an 'optimal' binding partner. Our results support the hypothesis that PDZ-peptide interactions may be best understood when one considers the sum of entropic and dynamic effects for each peptide as a whole entity, rather than preferences for specific residues at a given position.

Novel peptide recognized by RhoA GTPase

100%

Drulis-Fajdasz D., Jelen F., Oleksy A., Otlewski J.

2006

vol. 53

issue 3

515-524

A phage-displayed random 7-mer disulfide bridge-constrained peptide library was used to map the surface of the RhoA GTPase and to find peptides able to recognize RhoA switch regions. Several peptide sequences were selected after four rounds of enrichment, giving a high signal in ELISA against RhoA-GDP. A detailed analysis of one such selected peptide, called R2 (CWSFPGYAC), is reported. The RhoA - R2 interaction was investigated using fluorescence spectroscopy, chemical denaturation, and determination of the kinetics of nucleotide exchange and GTP hydrolysis in the presence of RhoA regulatory proteins. All measurements indicate that the affinity of the R2 peptide for RhoA is in the micromolar range and that R2 behaves as an inhibitor of: i) GDP binding to the apo form of RhoA (Mg2+- and nucleotide-free form of the GTPase), ii) nucleotide exchange stimulated by GEF (DH/PH tandem from PDZRhoGEF), and iii) GTP hydrolysis stimulated by the BH domain of GrafGAP protein.

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2 Acta Biochimica Polonica

1 2008

1 2006

2 Otlewski J.

1 Derewenda U.

1 Derewenda Z.

1 Drulis-Fajdasz D.

1 Jelen F.

1 Kasztura M.

1 Oleksy A.

1 Paduch M.

1 Smietana K.

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Degenerate specificity of PDZ domains from RhoA-specific nucleotide exchange factors PDZRhoGEF and LARG

Novel peptide recognized by RhoA GTPase