P. Cronet et al., INVESTIGATING THE STRUCTURAL DETERMINANTS OF THE P21-LIKE TRIPHOSPHATE AND MG2-SITE( BINDING), Journal of Molecular Biology, 249(3), 1995, pp. 654-664
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.