INVESTIGATING THE STRUCTURAL DETERMINANTS OF THE P21-LIKE TRIPHOSPHATE AND MG2-SITE( BINDING)

Amongst the superfamily of nucleotide binding proteins, the classical mononucleotide binding fold (CMBF), is the one that has been best char acterized structurally The common denominator of all the members is th e triphosphate/Mg2+ binding site, whose signature has been recognized as two structurally conserved stretches of residues: the Kinase 1 and 2 motifs that participate in triphosphate and Mg2+ binding, respective ly: The Kinase 1 motif is borne by a loop (the P-loop), whose structur e is conserved throughout the whole CMBF family; The low sequence simi larity between the different members raises questions about which inte ractions are responsible for the active structure of the P-loop. What are the minimal. requirements for the active structure of the P-loop? Why is the P-loop structure conserved despite the diverse environments in which it is found? To address this question, we have engineered th e Kinase 1 and 2 motifs into a protein that has the CMBF and no nucleo tide binding activity, the chemotactic protein from Escherichia coli, CheY. The mutant does not exhibit any triphosphate/Mg2+ binding activi ty. The crystal structure of the mutant reveals that the engineered P- loop is in a different conformation than that found in the CMBF. This demonstrates that the native structure of the P-loop requires external interactions with the rest of the protein. On the basis of an analysi s of the conserved tertiary contacts of the P-loop in the mononucleoti de binding superfamily, we propose a set of residues that could play a n important role in the acquisition of the active structure of the P-l oop.