Functional Roles of Loops 3 and 4 in the Cyclic Nucleotide Binding Domain of Cyclic AMP Receptor Protein from Escherichia coli

Abstract

Cyclic AMP is a ubiquitous secondary message that regulates a large variety of functions. The protein structural motif that binds cAMP is highly conserved with the exception of loops 3 and 4, whose structure and length are variable. The cAMP receptor protein of Escherichia coli, CRP, was employed as a model system to elucidate the functional roles of these loops. Based on the sequence differences between CRP and cyclic nucleotide gated channel, three mutants of CRP were constructed: deletion (residues 54 -56 in loop 3 were deleted), insertion (loop 4 was lengthened by 5 residues between Glu-78 and Gly-79) and double mutants. The effects of these mutations on the structure and function of CRP were monitored. Results show that the deletion and insertion mutations do not significantly change the secondary structure of CRP, although the tertiary and qua- Cyclic AMP serves as an intracellular message in both prokaryotes and eukaryotes by transmitting information through proteins such as protein kinase A (PKA) 1 , cyclic nucleotidegated ion channels (CNGC), and cAMP receptor protein in Escherichia coli (CRP). These proteins are involved in a very diverse set of cellular functions such as signal transduction, excitability, and gene expression (1-6). These proteins of diverse functions all consist of a cAMP binding motif. The structural motif, which serves as cAMP receptor, is found to display a high degree of similarity. X-ray crystallography and homology modeling results show that, despite obvious divergence of sequence among the receptor domains and significantly different biological functions of these proteins, their CNB domains appear to share a common architecture, all consisting of an ␣-helix (helix A), an eight-stranded ␤-roll, and two more ␣-helices (helices B and C). The body of the CNB pocket is mainly located in the ␤-roll, with the C-helix forming the back of the binding pocket (2,8). The superimposition of the structures of CNB domains from CRP and the regulatory subunits of PKA, as shown below in Fig. 1, indicates that the ␤-roll basically assumes the same structure with the exception of loops 3 and 4 between strands 4 and 5, and strands 6 and 7, respectively. In some cases, such as in CNGC and PKA, loop 3 is shortened whereas loop 4 is lengthened, as shown in Fig. 1. Only six residues (Gly-33, Gly-45, Gly-71, Glu-72, Arg-82, and Ala-84 using the CRP sequence as reference) are invariant among all members of the families. It has been suggested that the invariant residues play important and conserved roles in the folding and function of the CNB sites of these diverse proteins. Gly-33, Gly-45, and Gly-71 are involved in turns between strands of the ␤-roll; Arg-82 and Glu-72 contact the cyclic nucleotide, and the function of Ala-84 is uncertain (3). Despite large variation of primary sequences, the sizes of secondary structural elements of the CNB domain are much conserved among the family. For example, the alignment of CRP and CNGCs by keeping the six conserve residues at the same positions shows that the si...

cAMP Receptor Protein from <i>Escherichia coli</i> as a Model of Signal Transduction in Proteins – A Review

“…The main structural elements that are significantly different in the various proteins include loop 3 (formed by amino acids 50-61) and loop 4 (formed by amino acids 73-81) [Scott et al, 1996]. On the basis of screening mutations in the loops (deletion in loop 3, insertion in loop 4 and the combined mutations), it has been found that these loops can play important roles in the function of CRP [Chen and Ching Lee, 2003]. Loop 3 is involved in intersubunit signal communication, whereas loop 4 is involved in the cAMP-binding pocket and interdomain signal transmission [Shen et al, 2008].…”

The Secondary Structure of Calcineurin Regulatory Region and Conformational Change Induced by Calcium/Calmodulin Binding

“…For every point the fluorescence intensity was read at least 10 times to ensure that the result was not located at an extreme deviation region. Quenching data were plotted using the SternVolmer equation (21)…”

Structural Insights into the Mechanism of the Allosteric Transitions of Mycobacterium tuberculosis cAMP Receptor Protein

“…Deletion of Rv3676 results in impaired growth in laboratory medium, in bone marrow-derived macrophages, and in tubercle bacillus grown in a mouse model system (13). These observations suggest that Mtb-CRP plays a critical role in Mtb survival, especially in macrophages.Several models have been offered as possible explanations for how the sensory module can transmit the signal to the distant DNA-binding domain (9,(15)(16)(17). Perhaps the most intriguing question about the CRP family is how ligand binding within the effector domain can influence the properties of the DNA-binding domain.…”

“…Several models have been offered as possible explanations for how the sensory module can transmit the signal to the distant DNA-binding domain (9,(15)(16)(17). Perhaps the most intriguing question about the CRP family is how ligand binding within the effector domain can influence the properties of the DNA-binding domain.…”