Thermodynamic Analyses Reveal Role of Water Release in Epitope Recognition by a Monoclonal Antibody against the Human Guanylyl Cyclase C Receptor

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Methyltransferase-catalyzed modification of substrate DNA, RNA, or protein serves as a key signal in the regulation and maintenance of diverse biological processes in a range of organisms from prokaryotes to eukaryotes (1, 2). Subsequent to its isolation from Hemophilus haemolyticus nearly a quarter century ago, HhaI DNA methyltransferase (M.HhaI) 1 has been subjected to incisive investigations yielding detailed insights into the reaction chemistry (3-7). M.HhaI is an S-adenosyl-Lmethionine (AdoMet) dependent DNA methyltransferase that catalyzes the covalent attachment of a methyl group at the C-5 position of the aromatic ring of the first cytosine in the specific sequence 5Ј-GCGC-3Ј. Its ability to perform the methyl transfer within the B-DNA helix was rationalized by a localized "DNA backbone rotation" by nearly 180°(base flipping) without significantly bending or kinking of the rest of the duplex (8 -11). Base flipping serves to deliver the base into a concave catalytic site in the enzyme (8). The Gln 287 side chain fills up the gap generated concomitantly in the DNA bound with M.HhaI, leading to a stable and specific interruption of the aromatic -stacked base pairs, a feature recently utilized to evaluate long range electron migration through DNA (12). M.HhaI methylates all of the cytosines in poly(dG-dC) DNA (3), with hemimethylated DNA being the preferred substrate (13). Both the reaction product S-adenosyl-L-homocysteine (AdoHcy) (14) and, by implication, the cofactor AdoMet (7), appear to share the ability to drive M.HhaI to trap the target cytosine in the catalytic site to form a productive complex. These results portray AdoMet and AdoHcy as important modulators of the multiple binding modes of the DNA-bound enzyme.Despite a wealth of information on the structural, dynamic, and kinetic aspects of the M.HhaI mechanism, little attention has been paid to the energetics of AdoMet and AdoHcy recognition reactions. No molecule other than ATP serves as a cofactor in more diverse reactions than does AdoMet (15). It was, therefore, of interest to understand the thermodynamic basis of recognition of the ubiquitous cofactor AdoMet by methyltransferases, a feature shared among structurally related enzymes that catalyze reactions with diverse substrate specificities (16 -18). For example, a snapshot of a step that follows methyl transfer has been elucidated by crystallographic analyses of the DNA intermediate covalently bound to M.HhaI (8) and M.HaeIII (19). Despite its absence in the original crystallization mix of the ternary complex, it was AdoHcy that was found, fortuitously, to reside within the cofactor binding pocket (8) shared by AdoMet (16). This illustrates that the transfer of the methyl group from AdoMet to the target cytosine results in the formation not only of the modified (i.e. methylated) DNA but also the transformation of AdoMet into AdoHcy. On the other * This work was supported by grants from the Department of Biotechnology, Government of India (to D. N. R. and A. S.) and the Department of Scien...

Section: Cation-interaction Is Not Involved In the Origin Of Dual Modsupporting

confidence: 80%

“…AdoMet and AdoHcy solutions were then prepared with the final dialysate. Care was taken that the neutral solute osmolalities used were not significantly different from their ordinary molal concentrations (22)(23)(24)(25).…”

Section: Methodsmentioning

confidence: 99%

Water-assisted Dual Mode Cofactor Recognition by HhaI DNA Methyltransferase

Swaminathan¹,

Sankpal²,

Rao³

et al. 2002

Self Cite

“…This has been demonstrated for many biological systems, including complex formation between protein and DNA (48, 49), ferredoxin binding to ferredoxin-NADP ϩ reductase (50), and epitope recognition by a monoclonal antibody (51). A molecular reaction accompanied by a decrease in solvent-accessible surface is favored by decreasing the water activity.…”

Section: Resultsmentioning

confidence: 99%

Positive Contribution of Hydration on DNA Binding by E2c Protein from Papillomavirus

Lima

Silva

2004

Protein-nucleic acid interactions are responsible for the regulation of key biological events such as genomic transcription and recombination and viral replication. However, the recognition mechanisms involved in these processes are not completely understood. Here, we investigate the dominant forces involved in protein-protein and protein-DNA interactions for the 80-amino-acid C-terminal domain of the E2 protein (E2c) from human papillomavirus (HPV-16). The E2c protein is a homodimer that specifically binds to double-stranded DNA containing the consensus sequence ACCG-N 4 -CGGT, where N is any nucleotide. DNA binding affinity is reduced by lowering water chemical potential, accompanied by an increase in cooperativity. Wyman linkage relations between affinity and water chemical potential indicate that 11 additional water molecules are bound in the formation of the complex between E2c and DNA. Salt dissociation isotherms showed that 10 counterions are released upon association, even at low water activity, indicating that this latter variable does not change the electrostatic component of the interaction. Further analysis demonstrates a strong dependence of cooperativity of binding on the protein concentration. Altogether, these results reveal a novel binding pathway in which the consolidated complex may achieve its final form via a monomer-DNA intermediate, which favors the binding of a second monomer. This molecular mechanism reveals the contributions of multiple conformers in a tight virus genome modulation that seems to be important in the cell infection scenario. Human papillomavirus (HPV)1 is a small nonenveloped double-stranded DNA virus that causes epithelial lesions, which often develop into malignancies (1). The viruses HPV 16, -18, and -31 are denoted high risk HPV since they are directly related to cervical cancer. The regulation of gene expression in virus-infected cells is mediated by the factor E2, which controls the transcription of the HPV gene, including those related to malignant transformation. E2 can act either as an activator or as a repressor, depending on the alternate splicing of the product. Infected cells can be found containing (i) the C-terminal domain (E2c), responsible for DNA binding (2) and dimerization (3); (ii) the E2-E8 splicing product; and (iii) the full E2 protein (E2-full), consisting of the E2c, the N-terminal transactivation domain (E2TA), and an unstructured region with little known function. These three components recognize and bind specifically the palindromic sequence ACCGN 4 CGGT (2), where N is any base, although differences in affinity can arise depending on the N bases (4 -7). Part of the E2 structure has been solved: both for E2c, the DNA-binding domain (7-10), and for the transactivation domain (11,12).Much has appeared in the literature about the behavior of the E2c protein from the high risk HPV-16, in terms of homoassociation (13-16) and DNA binding and recognition, through equilibrium (6, 9, 14, 16 -20) and kinetic assays (4, 21). Although we have previously p...

“…This increase indicates that osmotic stress weakens the interaction between C3 and compstatin. Based on the water activity and solute osmolality, the following relationship can be used to calculate the change in the number of solute-excluded water molecules (⌬n w ) associated with the binding event (17). Plotting the logarithm of the association constant (K a ) against solute osmolality resulted in a slope of Ϫ0.17 for V4H9 and Ϫ0.12 for V4W/H9A binding.…”

Section: Effect Of Solvent On the C3-compstatinmentioning

confidence: 99%

“…In the case of proteinprotein interactions, water molecules act as molecular determinants of ligand recognition, thus altering the specificity of the interaction (15)(16)(17). To gain insight into the role of water molecules in the molecular recognition of compstatin by C3, we carried out osmotic stress experiments.…”

Section: Effect Of Solvent On the C3-compstatinmentioning

confidence: 99%

Thermodynamic Studies on the Interaction of the Third Complement Component and Its Inhibitor, Compstatin

Katragadda

Morikis

Lambris

2004

Compstatin is a 13-residue cyclic peptide that inhibits complement activation by binding to complement component, C3. Although the activity of compstatin has been improved severalfold using combinatorial and rational design approaches, the molecular basis for its interaction with C3 is not yet fully understood. In the present study, isothermal titration calorimetry was employed to dissect the molecular forces that govern the interaction of compstatin with C3 using four different compstatin analogs. Our studies indicate that the C3-compstatin interaction is an enthalpy-driven process. Substitution of the valine and histidine residues at positions 4 and 9 with tryptophan and alanine, respectively, resulted in the increase of enthalpy of the interaction, thereby increasing the binding affinity for C3. The data also suggest that the interaction is mediated by water molecules. These interfacial water molecules could be the source for unfavorable entropy and large negative heat capacity changes observed in the interaction. Although part of the negative heat capacity changes could be accounted for by the water molecules, the rest might be resulting from the conformational changes in C3 and/or compstatin up on binding. Finally, we propose based on the pK a values determined from the protonation studies that histidine on compstatin participates in protonation changes and contributes to the specificity of the interaction between compstatin and C3. These protonation changes vary significantly between the binding of different compstatin analogs to C3.