SMALL COMPOUNDS AND METABOLITES AS CONFORMATIONAL MODULATORS OF FULL-LENGTH PDK1

GROSS, LZF; HICKS, KG; SVERGUN, DI ; KLINKE, S; SACERDOTI, M; RUTTER, J; GHODE, A; BIONDI, RM; LEROUX, AE; GRAEWERT, MA; ANAND, GS

Mendoza

Congreso; LVI Reunión Científica anual SAIB y XV de SAMIGE; 2020

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

Protein kinases are veryimportant ON-OFF switches in cells. Dysregulation of their activities oftenlead to diseases, such as cancer or diabetes. Therefore, protein kinases aretightly and selectively regulated. Despite the large knowledge on the role of kinases,the structures of full-length protein kinases are vastly unknown and very fewstudies have investigated multidomain protein kinases in solution from astructural perspective. Phosphoinositide-dependent protein kinase 1 (PDK1) is a master AGC kinasethat phosphorylates at least other 23 AGC kinases, being PKB/Akt the mostrelevant substrate downstream of PI3-kinase, important for growth and cellsurvival and a drug target for cancer treatment. PDK1 consists of an N-terminalprotein kinase catalytic domain, followed by a linker and a C-terminal PHdomain. Over the years, our laboratory studied and characterized in detail the catalyticdomain of PDK1. In particular,we employed a chemical biology approach todescribe the allosteric communication between a hydrophobic regulatory site,termed PIF-pocket, and the ATP-binding site. We developed small compounds thatbind to the PIF-pocket regulatory site and allosterically affect theATP-binding site. We also described small compounds that bind at theATP-binding site and allosterically affect interactions at the regulatorysite.  Our previous studies opened thepossibility that metabolites binding at the ATP-binding site couldallosterically modulate protein kinase interactions and the formation ofprotein kinase complexes. This new regulation approach implies that the metabolicstate of cells could be linked to the regulation of cell signalling. Here Ipresent the crystal structure of the catalytic domain of PDK1 in complex with ametabolite bound to the ATP-binding site, DMP, and compare the structure of thecomplex and allosteric effects of this metabolite to the crystal structures andallosteric effects of the metabolites Adenine and Adenosine, which also bind atthe ATP-binding site. In addition to this, we identified small compounds thatcan modulate the conformation of full length PDK1. To study thisligand-dependent conformational modulation, we investigated the effect of thesesmall compounds on full length PDK1 using Hydrogen/Deuterium exchange studies aswell as small-angle X-ray scattering (SAXS) experiments. Finally, I willpresent our preliminary models on full length PDK1.