The X-ray crystal structure of the cAMP-liganded D138L mutant of Escherichia coli catabolite gene activator protein (CAP) was determined at a resolution of 1.66(A).This high resolution crystal structure reveals four cAMP binding sites in the CAP homodimer.Two anti-cAMPs locate between the β-barrel and the C-helix of each subunit; two syn-cAMPs bind on the surface of the C-terminal domain.With two syn-cAMP molecules bound, the D138L CAP becomes approximately symmetrical with both subunits assuming a "closed" conformation.In contrast to the WT CAP where backbone hydrogen bond is observed between D138 and G141 of loop3, in D138L, as loop3 is 3 amino acids shorter, the same structure is missing.Instead, a water molecule is seen hydrogen bondea to the amide nitrogen atom of L138 residue.Furthermore, the secondary structure alteration in the loop 3 provides an additional hydrogen bond in the β sheet which makes the cAMP binding domain more constrained.These changes make the hinge region of the mutant more flexible and susceptible to proteases.The results of protein dynamic experiments indicate that the structure of D138L mutant is more dynamic than that of WT, which may impact the recognition of specific DNA sequences.