ANATOMY OF PROTEIN POCKETS AND CAVITIES - MEASUREMENT OF BINDING-SITEGEOMETRY AND IMPLICATIONS FOR LIGAND DESIGN

Identification and size characterization of surface pockets and occlud ed cavities are initial steps in protein structure-based ligand design . A new program, CAST, for automatically locating and measuring protei n pockets and cavities, is based on precise computational geometry met hods, including alpha shape and discrete flow theory. CAST identifies and measures pockets and pocket mouth openings, as well as cavities. T he program specifies the atoms lining pockets, pocket openings. and bu ried cavities; the volume and area of pockets and cavities; and the ar ea and circumference of mouth openings. CAST analysis of over 100 prot eins has been carried out; proteins examined include a set of 51 monom eric enzyme-ligand structures, several elastase-inhibitor complexes, t he FK506 binding protein, 30 HIV-1 protease-inhibitor complexes, and a number of small and large protein inhibitors, Medium-sized globular p roteins typically have 10-20 pockets/cavities. Most often, binding sit es are pockets with 1-2 mouth openings; much less frequently they are cavities. Ligand binding pockets vary widely in size, most within the range 10(2)-10(3) Angstrom(3). Statistical analysis reveals that the n umber of pockets and cavities is correlated with protein size, but the re is no correlation between the size of the protein and the size of b inding sites. Most frequently, the largest pocket/cavity is thp active site, but there are a number of instructive exceptions. Ligand volume and binding site volume are somewhat correlated when binding site vol ume is less than or equal to 700 Angstrom(3), but the ligand seldom oc cupies the entire site. Auxiliary pockets near the active site have be en suggested as additional binding surface for designed ligands (Matto s C ct al., 1993, Nat Struct Biol 1:55-58). Analysis of elastase-inhib itor complexes suggests that CAST can identify ancillary pockets, suit able for recruitment in ligand design strategies. Analysis of the FK50 6 binding protein, and of compounds developed in SAR by NMR (Shuker SE et al.. 1996, Science 274:1531-1534), indicates that CAST pocket comp utation may provide a priori identification of target proteins for Lin ked-fragment design. CAST analysis of 30 HIV-1 protease-inhibitor comp lexes shows that the flexible active site pocket can vary over a range of 853-1,566 Angstrom(3), and that there are two pockets near or adjo ining the active site that may be recruited for ligand design.