Functional Roles of Loops 3 and 4 in the Cyclic Nucleotide Binding Domain of Cyclic AMP Receptor Protein from Escherichia coli
2003; Elsevier BV; Volume: 278; Issue: 15 Linguagem: Inglês
10.1074/jbc.m211551200
ISSN1083-351X
Autores Tópico(s)Biochemical and Structural Characterization
ResumoCyclic 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 quaternary structures are perturbed. The functional data indicate that loop 3 modulates the binding affinities of cAMP and DNA. Although the lengthened loop 4 may have some fine-tuning functions, the specific function of the original loop 4 of CRP remains uncertain. The function consequences of mutation in loop 3 of CRP are similar to that of site A and site B in the regulatory subunits of cyclic AMP-dependent protein kinases. Thus, the roles played by loop 3 in CRP may represent a more common mechanism employed by cyclic nucleotide binding domain in modulating ligand binding affinity and intramolecular communication. 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 quaternary structures are perturbed. The functional data indicate that loop 3 modulates the binding affinities of cAMP and DNA. Although the lengthened loop 4 may have some fine-tuning functions, the specific function of the original loop 4 of CRP remains uncertain. The function consequences of mutation in loop 3 of CRP are similar to that of site A and site B in the regulatory subunits of cyclic AMP-dependent protein kinases. Thus, the roles played by loop 3 in CRP may represent a more common mechanism employed by cyclic nucleotide binding domain in modulating ligand binding affinity and intramolecular communication. cAMP-dependent protein kinases cyclic AMP receptor protein cyclic nucleotide binding cyclic nucleotide-gated ion channels circular dichroism guanidine hydrochloride N-[4-[7-(diethylamino)-4-methylcoumarin-3-yl]phenyl] maleimide 8-anilino-1-naphthalene sulfonic acid 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 nucleotide-gated 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 (1Passner J.M. Schultz S.C. Steitz T.A. J. Mol. Biol. 2000; 304: 847-859Crossref PubMed Scopus (191) Google Scholar, 2Weber I.T. Shabb J.B. Corbin J.D. Biochemistry. 1989; 28: 6122-6127Crossref PubMed Scopus (90) Google Scholar, 3Shabb J.B. Corbin J.D. J. Biol. Chem. 1992; 267: 5723-5726Abstract Full Text PDF PubMed Google Scholar, 4Su Y. Dostmann R.G. Herberg F.W. Durick K. Xuong N.H. Ten Eyck L. Taylor S.S. Varughese K.I. Science. 1995; 269: 807-813Crossref PubMed Scopus (343) Google Scholar, 5Diller T.C. Madhusudan Xuong N.H. Taylor S.S. Structure. 2001; 9: 73-82Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 6Scott S.P. Harrison R.W. Weber I.T. Tanaka J.C. Protein Eng. 1996; 9: 333-344Crossref PubMed Scopus (29) Google Scholar). 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 α-helix (helix A), an eight-stranded β-roll, and two more α-helices (helices B and C). The body of the CNB pocket is mainly located in the β-roll, with the C-helix forming the back of the binding pocket (2Weber I.T. Shabb J.B. Corbin J.D. Biochemistry. 1989; 28: 6122-6127Crossref PubMed Scopus (90) Google Scholar, 8Weber I.T. Steitz T.A. Bubis J. Taylor S.S. Biochemistry. 1987; 26: 343-351Crossref PubMed Scopus (116) Google Scholar). 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 β-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 β-roll; Arg-82 and Glu-72 contact the cyclic nucleotide, and the function of Ala-84 is uncertain (3Shabb J.B. Corbin J.D. J. Biol. Chem. 1992; 267: 5723-5726Abstract Full Text PDF PubMed Google Scholar). 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 size differences in secondary structural elements are only located at two loops, e.g. loop 3 (between β4 and β5) of CNGCs is shorter than that in CRP by three residues, and loop 4 (between β6 and β7) of CNGCs is 5 residues longer than that of CRP (see Fig. 2) (6Scott S.P. Harrison R.W. Weber I.T. Tanaka J.C. Protein Eng. 1996; 9: 333-344Crossref PubMed Scopus (29) Google Scholar). Similarly, loops 3 and 4 among the various protein kinase isozymes also show heterogeneity in size, as shown in Fig. 2.Figure 2Amino acid sequence alignment of CRP and other cyclic nucleotide binding domains. See Refs. 6Scott S.P. Harrison R.W. Weber I.T. Tanaka J.C. Protein Eng. 1996; 9: 333-344Crossref PubMed Scopus (29) Google Scholar and 8Weber I.T. Steitz T.A. Bubis J. Taylor S.S. Biochemistry. 1987; 26: 343-351Crossref PubMed Scopus (116) Google Scholar for citations of specific protein sequences.View Large Image Figure ViewerDownload (PPT)Based on the sequence alignment, it is interesting to note that loops 3 and 4 are the only structural elements that are different in size among these various sources of CNB domain. To elucidate the roles of these loops the cAMP receptor protein, CRP, from E. coli is employed as a model system for investigation. CRP of Escherichia coli, also referred to as the catabolite gene activator protein, is a 47,238-Da homodimer. Each subunit has two domains: the large N-terminal domain is a cyclic nucleotide binding domain, and the small C-terminal domain is a DNA binding domain. ApoCRP has very low affinity for DNA and cannot differentiate between specific and nonspecific DNA sequences, whereas holoCRP exhibits high affinity for specific DNA sequences. It is known that binding of cAMP allosterically induces CRP to assume conformations that exhibit high affinity for specific DNA sequences (9Harman J.G. Biochim. Biophys. Acta. 2001; 1547: 1-17Crossref PubMed Scopus (208) Google Scholar, 10Kolb A. Busby S. Buc H. Garges S. Adhya S. Annu. Rev. Biochem. 1993; 62: 749-795Crossref PubMed Google Scholar, 11Busby S. Ebright R.H. J. Mol. Biol. 1999; 293: 199-213Crossref PubMed Scopus (632) Google Scholar, 12Heyduk T. Lee J.C. Biochemistry. 1989; 28: 6914-6924Crossref PubMed Scopus (115) Google Scholar, 13Passner T.M. Steitz T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2843-2847Crossref PubMed Scopus (158) Google Scholar). It has been suggested that allosteric conformational change, which includes subunit realignment and domain rearrangement, occurs upon the binding of cAMP to CRP. These changes are mediated by interactions involving the subunit and domain interfaces. The C-helix and the hinge region between the domains have been found to play key roles in transmitting the allosteric signal. Although earlier spectroscopic comparison between the holo- and apoCRP showed no apparent secondary structural changes (14DeGrazia H. Harman J.G. Tan G.S. Wartell R.M. Biochemistry. 1990; 29: 3557-3562Crossref PubMed Scopus (22) Google Scholar, 15Tan G.S. Kelly P. Kim J. Wartell R.M. Biochemistry. 1991; 30: 5076-5080Crossref PubMed Scopus (25) Google Scholar, 16Heyduk E. Heyduk T. Lee J.C. J. Biol. Chem. 1992; 267: 3200-3204Abstract Full Text PDF PubMed Google Scholar), the results of protein footprinting experiments and recent NMR studies indicate that there are wide-ranging structural differences between apoCRP and holoCRP. cAMP binding, while perturbing the β-roll that forms the cAMP binding pocket, has little effect on the secondary structure elements contained in either the N- or the C-terminal domains. There does, however, appear to be a significant difference around the C terminus of C-helix, the hinge region, and loop 3 (1Passner J.M. Schultz S.C. Steitz T.A. J. Mol. Biol. 2000; 304: 847-859Crossref PubMed Scopus (191) Google Scholar, 17Baichoo N. Heyduk T. Biochemistry. 1997; 36: 10830-10836Crossref PubMed Scopus (46) Google Scholar, 18Baichoo N. Heyduk T. Protein Sci. 1999; 8: 518-528Crossref PubMed Scopus (40) Google Scholar, 19Won H.S. Yamazaki T. Lee T.W. Yoon M.K. Park S.H. Otoma T. Kyogoku Y. Lee B.J. Biochemistry. 2000; 39: 13953-13962Crossref PubMed Scopus (57) Google Scholar, 20Won H.S. Lee T.W. Park S.H. Lee B.J. J. Biol. Chem. 2002; 277: 11450-11455Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). These studies identified the location of structural changes induced by cAMP binding but do not provide information on the role of these structures in this β-roll motif in binding cyclic nucleotides.It was postulated that loop 3 in CRP is involved in both interdomain and intersubunit interactions, whereas loop 4 contacts the coiled-coil C-helices and forms part of the dimer interface (1Passner J.M. Schultz S.C. Steitz T.A. J. Mol. Biol. 2000; 304: 847-859Crossref PubMed Scopus (191) Google Scholar), although the effects of these interactions in CRP function were not predictable by structural analysis alone. In recent studies, it was shown that a D53H mutation in loop 3 leads to enhancements of the magnitude of positive cooperativity in cAMP binding and affinity for specific DNA (21Lin S.H. Kovac L. Chin A.L. Chin C.C.Q. Lee J.C. Biochemistry. 2002; 41: 2946-2955Crossref PubMed Scopus (26) Google Scholar, 22Lin S.H. Lee J.C. Biochemistry. 2002; 41: 11857-11867Crossref PubMed Scopus (54) Google Scholar). These solution biophysical data are consistent with the proposal that loop 3 plays a role in interdomain and intersubunit communications, although the specific nature of this role is unknown. In this study, three mutants of CRP are constructed according to the difference in sequence alignment between CRP and CNGC, namely, a deletion of residues 54–56 in loop 3 and insertion of 5 residues between residues 78 and 79, respectively. The choices for specific sequences for deletion and insertion are based on the availability of information on CNGC. Consequently, it is possible to compare and contrast the data acquired in this study with the literature. These mutants are the subjects of investigation to elucidate the roles of these loops in the normal function of CRP.MATERIALS AND METHODSAll in vitro experiments were conducted in TEK (100) buffer (50 mm Tris-HCl, 100 mm KCl, and 1 mm EDTA at pH 7.8 and 22.5 °C). The concentrations of protein, cyclic nucleotides, and fluorescence probes were determined by absorption spectroscopy using the following absorption coefficients: 40,800 m−1cm−1 at 278 nm for CRP and its mutants, 14,650 m−1cm−1at 271 nm for cAMP, 12,950 m−1cm−1 at 254 nm for cGMP, 33,000m−1cm−1 at 385 nm for CPM, and 6,240 m−1cm−1 at 351 nm for ANS. All the solutions were made with reagent grade or higher grade chemicals and filtered prior to use.Site-directed MutagenesisThe sites and nature of mutations in loops 3 and 4 are based on the sequence differences between CRP and CNGC, namely, a deletion of Glu-54, Glu-55, and Gly-56 in loop 3 and an insertion of the sequence KGSKM between Glu-78 and Gly-79 in loop 4, as shown in Fig. 2. An overlap extension PCR method was used (23Sambrook J. Russel D.W. 3rd Ed. Molecular Cloning: A Laboratory Manual. 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001: 13.36-13.39Google Scholar). The outer pairs of primers include anNdeI site and an HindIII site, respectively. The sequences were: TAA CCG CATATG GTG CTT GG and CCA CTC CGA CAAGCTT AA CGA GTG CCG. The sequences of mutagenesis primers for insertion were ACG TTC CTG GCC CATCTTAGAGCCCTT CTC TTC AAA CAG GCC CAG and GTT TGA AGA G AAGGGCTCTAAGATG GGC CAG GAA CGT AGC GCA; those for deletion were AGG AGA GGA TCA TTT CTT T GTC TTT GAT CAG CAC TGC C and GGC AGT GCT GAT CAA AGA C AAA GAA ATG ATC CTC TCC T. The products of the second round of amplification were inserted into the pET30a plasmid, and the constructs were sequenced after cloning.Protein PurificationWild-type and mutant CRPs were purified from E. coli strain HMS174(DE3) using a previously described protocol (21Lin S.H. Kovac L. Chin A.L. Chin C.C.Q. Lee J.C. Biochemistry. 2002; 41: 2946-2955Crossref PubMed Scopus (26) Google Scholar, 24Cheng X. Kovac L. Lee J.C. Biochemistry. 1995; 34: 10816-10826Crossref PubMed Scopus (38) Google Scholar). All purified CRP proteins were >99% homogeneous as judged by SDS-PAGE stained by Coomassie Blue; 50–60 μg of CRP was routinely loaded onto each lane. Furthermore, the ratios of the absorbance at 280 nm to that at 260 nm were >1.86, indicating the absence of nucleic acid contamination. The mass of proteins was further checked by mass spectrometry.Analytical UltracentrifugationExperiments were conducted at appropriate speeds in a Beckman Optima XLA analytical ultracentrifuge equipped with absorbance optics and an An60Ti rotor. Sedimentation velocity experiments were performed at 42K rpm. Velocity data were collected at 280 nm at a spacing of 0.002 cm with no averaging in a continuous scan mode and were analyzed using DCDT+ version 1.12. The reported weight-average sedimentation coefficient values (s̄20,w) obtained from DCDT+ were calculated by a weighted integration over the entire range of sedimentation coefficients covered by the g(s) distribution and corrected for the solution density and viscosity.The apparent weight-average molecular weights were obtained by fitting the sedimentation equilibrium data with the following equation,C=E+C1exp(1−ν¯ρ)ω22RTM(r2−ro2)Equation 1 +C22Kaexp(1−ν¯ρ)ω22RT2M(r2−ro2)where C is the observed CRP concentration in absorbance at radial position r, E is the baseline offset, C1 and C2 are the CRP concentrations of monomeric and dimeric CRP, respectively, at the meniscus r0,ν̄ is the partial specific volume, ρ is the solvent density, ω is the angular velocity, M is the apparent weight-average molecular weight, and R and T are the gas constant and temperature in degrees kelvin, respectively.Ka is the apparent association constant. The value of ν̄ of CRP in Tris buffer is 0.744 and was derived from the amino acid composition of CRP using the method of Cohn and Edsall (25Cohn E.J. Edsall J.T. Proteins, Amino Acids and Peptides. Van Nostrans-Reinhold, NJ1943: 372Google Scholar). The apparent partial specific volumes of wild-type and mutant CRPs in 6m GdnHCl were calculated using the procedure of Lee and Timasheff (26Lee J.C. Timasheff S.N. Methods Enzymol. 1979; 61: 49-57Crossref PubMed Scopus (45) Google Scholar). The corresponding values in lower concentrations of GdnHCl were interpolated by assuming a linear relationship between GdnHCl bound and denaturant concentration.The quaternary structure of CRP mutant was monitored by sedimentation equilibrium using a published procedure (27Cheng X. Lee J.C. Biochemistry. 1998; 37: 51-60Crossref PubMed Scopus (15) Google Scholar). In most cases, the subunit-subunit interaction of CRP was strong and could not be estimated directly by sedimentation equilibrium. The CRP dimerization was weakened by increasing amounts of GdnHCl, and the quaternary structure of CRP was monitored under each GdnHCl concentration. The loading CRP concentrations were between 0.2 and 0.4 mg/ml. Usually, 200-μl samples were loaded in a 12-mm Epon charcoal-filled centerpiece. The high speed, meniscus depletion procedure was employed (28Yphantis D.A. Biochemistry. 1964; 3: 297-317Crossref PubMed Scopus (2018) Google Scholar).Having determined the value of Ka by Equation 1, ΔGa values, the free energy changes for subunit assembly at different concentrations of GdnHCl, were calculated and fitted by a linear least-squares analysis to Equation 2,−RT lnKa=ΔGa=ΔGao+ma[GdnHCl]Equation 2 where R and T are the gas constant and absolute temperatures, respectively, ΔGaois the extrapolated free energy changes of subunit assembly of CRP in buffer, and ma is the dependence of ΔGa on denaturant concentration.Circular Dichroism Data Acquisition and AnalysisCD measurements were performed on an AVIV 62DS CD spectropolarimeter using a 0.1-cm (for far-UV region) or 1.0-cm (for near-UV region) path length microcuvette (200-μl capacity). The protein concentration used was 7 μm. CD spectra were measured over the range of 200–320 nm. Each spectrum was recorded in 0.5-nm wavelength increments, and signal was acquired for 1 s at each wavelength. Each measurement was performed in triplicate. Deviations between scans were negligible. Baseline subtraction and smoothing of spectra curves were performed using the AVIV CDS program.Fluorescence Data Acquisition and AnalysisFluorescence measurements were carried out in 1-cm quartz cuvettes at 22.5 °C using a PerkinElmer Life Sciences LS50B luminescence spectrometer. Protein concentration was 5 μm. Samples were excited at 295 nm, and tryptophan emission was monitored from 310 to 400 nm. Acrylamide quenching measurements were carried out on samples containing acrylamide (0–0.7 m), either without or with 200 μm cAMP. Quenching data were plotted using the Stern-Volmer equation,Fo/F=(1+Ksv[Q])(1+V[Q])Equation 3 where Fo/F is the fractional decrease in fluorescence due to the quencher ([Q]), and Ksv and V are the collisional and static quenching constants, respectively.Cyclic Nucleotide Binding AssayCyclic nucleotide binding to CRP and mutants were measured by the quenching of ANS-CRP fluorescence according to the protocol described previously with minor modification (12Heyduk T. Lee J.C. Biochemistry. 1989; 28: 6914-6924Crossref PubMed Scopus (115) Google Scholar). Protein concentration was 12.5 μm. The binding data fitted with a three-site model (for cAMP binding to wild-type CRP) and a two-site model (for cAMP binding to mutants and all cGMP binding), respectively, in accordance to a previous observation (22Lin S.H. Lee J.C. Biochemistry. 2002; 41: 11857-11867Crossref PubMed Scopus (54) Google Scholar),CRP+L↔k1CRP·L+L↔k2CRP·L2+L↔k3CRP·L3Equation 4 where Ki is the association constant for the binding of the ith ligand. The observed fluorescence parameter is related to Ki by,Fobs=∑inXiFi;Xi=αi/∑i=0nαiEquation 5 where Fobs is the normalized value of observed fluorescence intensity at 480 nm, n is the total number of L molecules bound to a CRP molecule, iis the number of bound L molecules, and Xi and Fi are the fractions of CRP sub-conformation with different numbers of L bound and its fluorescence property, respectively. Xi is related to the fraction distribution parameter, αi, which corresponds to the number of L bound: for i = 0, α0 = 1; i = 1, α1 = 2k1[L];i = 2, α2 =k1k2[L]2; and i = 3, α3 = 2k1k2k3[L]3.DNA BindingFluorescence anisotropy measurements, by the SLM 8000C spectrofluorometer, were employed for quantitative evaluation of the CRP-DNA interaction. The DNA binding site was the 26-bp fragment of the lac PI promoter with the sequence 5′-ATTAATGTGAGTTAGCTCACTCATTA-3′. The underlined sequence is the primary binding site for CRP. The reaction mixture of 1300–1350 μl contained 12 nm of the CPM-labeled 26-bp fragment of lac promoter DNA and 230 μm cyclic nucleotide. At 230 μm, the high affinity sites for cyclic nucleotides are occupied in all CRPs employed in this study (22Lin S.H. Lee J.C. Biochemistry. 2002; 41: 11857-11867Crossref PubMed Scopus (54) Google Scholar). The detailed experimental and data analysis protocols have been previously described (21Lin S.H. Kovac L. Chin A.L. Chin C.C.Q. Lee J.C. Biochemistry. 2002; 41: 2946-2955Crossref PubMed Scopus (26) Google Scholar). Briefly, the data were fitted to the following equation by non-linear least-squares to determine the apparent association constant for CRP-DNA interaction,K,A=AD+(APD−AD)×(KDT+KPT)+1−(KDT+KPT+1)2−4K2DTPT2KDTEquation 6 where A is the measured value of the anisotropy,AD and APD are values of anisotropy with free DNA and CRP·DNA complex, respectively,DT and PT are the total molar concentrations of DNA and dimeric protein, respectively.GdnHCl DenaturationStock solutions of 6.9 mGdnHCl were prepared in TEK (100), and the concentrations were determined with a Mettler-Paar Precision density meter. Proteins at 5 μm were unfolded in various concentrations of GdnHCl for 1 h at room temperature. Protein unfolding was monitored by CD, and the data were expressed as SD, the measured CD signal was normalized to SD at 0m GdnHCl.DISCUSSIONSome loops in proteins seem to serve as no more than connectors between secondary structural elements, others play much more important functional roles, such as in defining stability (29Thompson M.J. Eisenberg D. J. Mol. Biol. 1999; 290: 595-604Crossref PubMed Scopus (246) Google Scholar, 30Minard P. Scalley-Kim M. Watters A. Baker D. Protein Sci. 2001; 10: 129-134Crossref PubMed Scopus (21) Google Scholar). On the basis of structural information and results of mutagenesis analyses, it was proposed that loop 3 in CRP is involved in both interdomain and intersubunit interactions, whereas loop 4 contacts the C-helices and is proposed to form part of the dimer interface (1Passner J.M. Schultz S.C. Steitz T.A. J. Mol. Biol. 2000; 304: 847-859Crossref PubMed Scopus (191) Google Scholar). Some point mutations, either within or just outside of these loops, have been reported to significantly affect the function of CRP, e.g. K52N, D53H, and S62F (beside loop 3) and E72A, K82Q, and S83K (beside loop 4) (21Lin S.H. Kovac L. Chin A.L. Chin C.C.Q. Lee J.C. Biochemistry. 2002; 41: 2946-2955Crossref PubMed Scopus (26) Google Scholar, 22Lin S.H. Lee J.C. Biochemistry. 2002; 41: 11857-11867Crossref PubMed Scopus (54) Google Scholar, 31Gronenborn A.M. Sandulache R. Gartner S. Clore G.M. Biochem. J. 1988; 253: 801-807Crossref PubMed Scopus (18) Google Scholar, 32Chu S.Y. Tordova M. Gilliland G.L. Gorshkova I. Shi Y. Wang S. Schwarz F.P. J. Biol. Chem. 2001; 276: 11230-11236Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 33Moore J. Kantorow M. Vanderzwaag D. McKenney K. J. Bacteriol. 1992; 174: 8030-8035Crossref PubMed Google Scholar, 34Lee E.J. Glasgow J. Leu S.F. Belduz A.O. Harman J.G. Nucleic Acids Res. 1994; 22: 2894-2901Crossref PubMed Scopus (44) Google Scholar). Results from protein footprinting and NMR experiments indicate that the regions, including these loops exhibit significant environmental changes upon cAMP binding (18Baichoo N. Heyduk T. Protein Sci. 1999; 8: 518-528Crossref PubMed Scopus (40) Google Scholar, 19Won H.S. Yamazaki T. Lee T.W. Yoon M.K. Park S.H. Otoma T. Kyogoku Y. Lee B.J. Biochemistry. 2000; 39: 13953-13962Crossref PubMed Scopus (57) Google Scholar). All these results imply that these loops are involved in the functioning of CRP. This conjecture on structure-function correlation is further supported by an alignment analysis of the protein sequence of CNB (Fig. 2) that reveals an intriguing pattern. The sizes of loops 3 and 4 are the only ones of the loop structures that are varied. In cyclic nucleotide-gated channels there are deletion and extension in the sequences of loop 3 and 4, respectively. In cyclic nucleotide-dependent protein kinases, the sequence variation mostly resides in loop 3. Thus, it is of interest to probe for the roles of these loops in CRP.CRP is a transcription factor that exhibits allosteric behavior. There is homotropic effects in the biding of cAMP and heterotropic effect between cAMP and DNA bindings. The deletion of residues 54–56 (EEG) in loop 3 leads to the most significant perturbations in the normal functional properties of CRP. The binding affinity of the first cAMP molecule is significantly weaker than that of the wild-type CRP, however, the mutation also enhances the binding affinity for the second cAMP molecule, leading to a significant enhancement of positive cooperativity as indicated by the steeper binding isotherm (Fig. 9 and Table II). These results indicate that the deletion of residues 54–56 alters the site-site interactions in cAMP binding, leading to an enhancement of the homotropic effect. In addition, the deletion mutation leads to a detectable, albeit small, decrease in DNA affinity,i.e. a negative impact on the heterotropic effect between cAMP and DNA binding sites (Fig. 10). This mutation also lowers the energetics of subunit assembly. Thus, a perturbation of this loop amplifies its impact on functional sites that are located at different parts of CRP and spatially quite a few angstroms away. The specific nature of functional impacts by loop 3 mutation on CRP is apparently also observed in the various isozymes of protein kinases. As shown in Figs. 1 and 2, the sequence differences between sites A and B of the regulatory subunit are often localized in loop 3, namely, in general there is a deletion of a few residues in loop 3 of site A as compared with site B. It has long been established that the binding affinity of site A is lower than site B, an observation parallel that of the deletion and wild-type CRP, respectively. It is not surprising that cooperativity of ligand binding is different between these two type of sites (35Corbin J.D. Sugden P.H. West L. Flockhart D.A. Lincoln T.M. McCarty D. J. Biol. Chem. 1978; 253: 3997-4003Abstract Full Text PDF PubMed Google Scholar, 36Ogreid D. Doskeland S.O. FEBS Lett. 1981; 129: 287-292Crossref PubMed Scopus (57) Google Scholar, 37Ogreid D. Doskeland S.O. FEBS Lett. 1982; 150: 161-166Crossref PubMed Scopus (33) Google Scholar, 38Ogreid D. Doskeland S.O. Miller J.P. J. Biol. Chem. 1983; 258: 1041-1049Abstract Full Text PDF PubMed Google Scholar, 39Robinson-Steiner A.M. Corbin J.D. J. Biol. Chem. 1983; 258: 1032-1040Abstract Full Text PDF PubMed Google Scholar, 40Doskeland S.O. Ogreid D. J. Biol. Chem. 1984; 259: 2291-2301Abstract Full Text PDF PubMed Google Scholar, 41Bubis J. Taylor S.S. Biochemistry. 1987; 26: 3478-3486Crossref PubMed Scopus (25) Google Scholar, 42Herberg F.W. Taylor S.S. Dostmann W.R.G. Biochemistry. 1996; 35: 2934-2942Crossref PubMed Scopus (106) Google Scholar).One might speculate on the mechanism that enables loop 3 to exert these homotropic and heterotropic effects. Residue 136 of the adjacent subunit forms a complex with loop 3. This might be one of the paths of intersubunit and interdomain interactions. Any mutation that leads to a perturbation of this interaction could be expected to affect the allosteric behavior, as shown in this study. Furthermore, the nature of the perturbation could be manifested to yield different functional consequences. For example, mutations at residues 52 and 62 lead to decreases in both homotropic and heterotropic effects, but mutation at residues 53 leads to an opposite effect (21Lin S.H. Kovac L. Chin A.L. Chin C.C.Q. Lee J.C. Biochemistry. 2002; 41: 2946-2955Crossref PubMed Scopus (26) Google Scholar, 22Lin S.H. Lee J.C. Biochemistry. 2002; 41: 11857-11867Crossref PubMed Scopus (54) Google Scholar). Thus, apparently, perturbations of residues in loop 3 can modulate the allosteric effects either positively or negatively. Therefore, the role of loop 3 is a modulator in the true sense.The insertion of five residues in loop 4 seems to have only marginal effects on the functional properties of CRP. There is no significant perturbation in cAMP binding, and the effect on DNA binding is small, although the energetics of subunit assembly is reduced. Thus, these results are consistent with the proposal that loop 4 forms part of the dimer interface (1Passner J.M. Schultz S.C. Steitz T.A. J. Mol. Biol. 2000; 304: 847-859Crossref PubMed Scopus (191) Google Scholar). Results of the double mutant show that
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