ARCHITECTURE AND INTRASTERIC REGULATION OF GIANT PROTEIN KINASES. B. Kobe, J. Heierhorst, S.C. Feil, M.W. Parker, G.M. Benian* and B.E. Kemp. St. Vincent's Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia; *Dept. of Pathology, Emory University, Atlanta, Georgia 30322, USA.

Protein kinases are key regulatory enzymes in most cellular processes and must themselves be tightly regulated. Such regulation is often achieved by an intrasteric mechanism, where a part of the enzyme binds to the active site and autoinhibits its activity. The giant myosin associated proteins twitchin, projectin and titin are the largest members of the protein kinase family (600-3000 kDa) and have important roles in muscle contraction and cytoskeletal structure. Their sequences consist of numerous fibronectin type III-like and immunoglobulin (Ig)-like motifs, and a single autoinhibited protein Ser/Thr kinase structurally related to vertebrate myosin light chain kinases. We have recently shown that twitchin can be activated by the Ca²⁺/S100A1₂ protein complex. To study the regulatory mechanisms of giant protein kinases, we determined the crystal structures of two recombinant fragments of twitchin. The structure of the autoinhibited twitchin kinase from Aplysia (A) reveals the conservation and flexibility of the autoinhibitory mechanism in giant protein kinases. The autoinhibitory sequence is positioned in the cleft between the two protein kinase lobes and inhibits the kinase by extensive contacts with residues implicated in substrate binding, ATP binding and catalysis. The structure of an extended fragment of C. elegans twitchin (B) containing the autoinhibited kinase domain and a C-terminal Ig-like domain shows that the Ig-like domain extends from the kinase domain opposite to the active site and exposes possible myosin-interacting surfaces. Our studies of the regulation and domain architecture of giant protein kinases jointly with an analysis of autoinhibitory sequences of other autoinhibited kinases point to possible common and diverse features of the intrasteric regulatory mechanisms.