Abstract
The crystal structure of cyclic AMP-catabolite activator protein (CAP) from Escherichia coli containing cobalt(II) chloride and ammonium sulfate is reported at 1.97 Å resolution. Each of the two CAP subunits in the asymmetric unit binds one cobalt(II) ion, in each case coordinated by N-terminal domain residues His19, His21 and Glu96 plus an additional acidic residue contributed via a crystal contact. The three identified N-terminal domain cobalt-binding residues are part of a region of CAP that is important for transcription activation at class II CAP-dependent promoters. Sulfate anions mediate additional crystal lattice contacts and occupy sites corresponding to DNA backbone phosphate positions in CAP-DNA complex structures.
Highlights
catabolite activator protein (CAP) is a DNA-binding protein and transcription activator that recruits RNA polymerase to more than 100 genes in Escherichia coli (Busby & Ebright, 1999; Lawson et al, 2004; Zheng et al, 2004)
We describe here the structure of cAMP–CAP in a novel fourth crystal form in which cobalt(II) binds to key residues of CAP activating region 2 (AR2)
Our efforts to characterize a CAP–DNA complex fortuitously yielded a crystal structure for cAMP–CAP without DNA in a novel crystal form in which cobalt and sulfate ligands are bound to CAP
Summary
CAP (catabolite activator protein) is a DNA-binding protein and transcription activator that recruits RNA polymerase to more than 100 genes in Escherichia coli (Busby & Ebright, 1999; Lawson et al, 2004; Zheng et al, 2004). The CAP dimer consists of two identical 209-residue subunits, each with an N-terminal binding domain for allosteric effector cAMP, and a C-terminal winged helix–turn–helix DNA-binding motif. CAP has three defined ‘activating regions’, patches of surface-exposed residues that interact with RNA polymerase (RNAP) in specific CAP-dependent promoter classes. AR2, located on the N-terminal cAMP-binding domain, functions at class II promoters and is predicted to make direct contact with an acidic loop of the RNAP I subunit Nterminal domain ( NTDI; Niu et al, 1996; Busby & Ebright, 1999; Lawson et al, 2004). AR3 is located on the N-terminal domain and is predicted to make direct contact with RNAP subunit region 4 (Rhodius & Busby, 2000)
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