STRUCTURE OF HUMAN CYCLIN-DEPENDENT KINASE INHIBITOR P19(INK4D) - COMPARISON TO KNOWN ANKYRIN-REPEAT-CONTAINING STRUCTURES AND IMPLICATIONSFOR THE DYSFUNCTION OF TUMOR-SUPPRESSOR P16(INK4A)

Background: The four members of the INK4 gene family (p16(INK4a), P15( INK4b), p18(INK4c) and p19(INK4d)) inhibit the closely related cyclin- dependent kinases CDK4 and CDK6 as part of the regulation of the G(1)- ->S transition in the cell-division cycle. Loss of INK4 gene product f unction, particularly that of p16(INK4a), is found in 10-60% of human tumors, suggesting that broadly applicable anticancer therapies might be based on restoration of p16(INK4a) CDK inhibitory function. Althoug h much less frequent, defects of p19(INK4d) have also been associated with human cancer (osteosarcomas). The protein structures of some INK4 family members, determined by nuclear magnetic resonance (NMR) spectr oscopy and X-ray techniques, have begun to clarify the functional role of p16(INK4a) and the dysfunction introduced by the mutations associa ted with human tumors. Results: The crystal structure of human p19(INK 4d) has been determined at 1.8 Angstrom resolution using multiple isom orphous replacement methods. The fold of p19(INK4d) produces an oblong molecule comprising five approximately 32-residue ankyrin-like repeat s. The architecture of the protein demonstrates the high structural si milarity within the INK4 family. Comparisons to other ankyrin-repeat-c ontaining proteins (GABP beta, 53BP2 and myotrophin) show similar stru ctures with comparable hydrogen-bonding patterns and hydrophobic inter actions. Such comparisons highlight the splayed P-loop geometry that i s specific to INK4 inhibitors. This geometry is the result of a modifi ed ankyrin structure in the second repeat. Conclusions: Among the INK4 inhibitors, the highest amino acid sequence conservation is found in the helical stacks; this conservation creates a conserved P-loop geome try specific to INK4 inhibitors. Therefore, in addition to models whic h predict that the conserved helix alpha 6 is responsible for CDK inhi bition, a binding mode whereby the loops of INK4 proteins bind to the CDKs should also be considered. A similar loop-based interaction is se en in the complex formed between the ankyrin-repeat-containing protein GABP beta and GABP alpha. This mode of binding would be consistent wi th the observation that p16(INK4a) is sensitive to deleterious mutatio ns found throughout this tumor suppressor protein; these mutations pro bably destabilize the three-dimensional structure.