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Subunit interacting surfaces of human haemoglobin. Localization of the alpha-subunit-beta-subunit interacting surfaces on the beta-chain by a comprehensive synthetic strategy.

ABSTRACT: A synthetic approach is introduced for localization of subunit interacting surfaces in oligomeric proteins. It consists of studying the binding activity of consecutive uniform overlapping peptides encompassing an entire subunit to the other, radiolabelled, subunit. This permits the establishment of the full profile of peptides that bind the other intact subunit. This approach has been demonstrated with haemoglobin, and its application here with the beta-chain peptides has enabled the localization on the beta-chain of the submolecular regions responsible for its binding to alpha-chain in solution. There was good agreement between the binding surfaces found here in solution and those expected from the crystal structure. There were also, however, some significant differences in the levels of binding found in solution and those expected from the crystal. Peptide 21-35 possessed much higher binding activity than would be expected from its contribution to subunit association in the crystal. Conversely, other regions expected to possess considerable binding capacity for alpha-chain either showed low (peptides 111-125 and 121-135) or almost no binding (peptides 91-105 and 101-115) capacity. On the other hand, two interacting surfaces (within peptides 11-25 and 71-85) that make a contribution in solution do not appear to play a role in the crystal. It is concluded that the regions of subunit association in solution are close to, but not identical with, those in the crystal. The approach should serve as an effective method for localization of subunit interacting surfaces of unknown proteins, even those that can be isolated only in traces.

SUBMITTER: Yoshioka N

PROVIDER: S-EPMC1146587 | biostudies-other | 1986 Mar

REPOSITORIES: biostudies-other

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Project description:Abasic (AP) sites in DNA arise through both endogenous and exogenous mechanisms. Since AP sites can prevent replication and transcription, the cell contains systems for their identification and repair. AP endonuclease (APEX1) cleaves the phosphodiester backbone 5' to the AP site. The cleavage, a key step in the base excision repair pathway, is followed by nucleotide insertion and removal of the downstream deoxyribose moiety, performed most often by DNA polymerase beta (pol-beta). While yeast two-hybrid studies and electrophoretic mobility shift assays provide evidence for interaction of APEX1 and pol-beta, the specifics remain obscure. We describe a theoretical study designed to predict detailed interacting surfaces between APEX1 and pol-beta based on published co-crystal structures of each enzyme bound to DNA. Several potentially interacting complexes were identified by sliding the protein molecules along DNA: two with pol-beta located downstream of APEX1 (3' to the damaged site) and three with pol-beta located upstream of APEX1 (5' to the damaged site). Molecular dynamics (MD) simulations, ensuring geometrical complementarity of interfaces, enabled us to predict interacting residues and calculate binding energies, which in two cases were sufficient (approximately -10.0 kcal/mol) to form a stable complex and in one case a weakly interacting complex. Analysis of interface behavior during MD simulation and visual inspection of interfaces allowed us to conclude that complexes with pol-beta at the 3'-side of APEX1 are those most likely to occur in vivo. Additional multiple sequence analyses of APEX1 and pol-beta in related organisms identified a set of correlated mutations of specific residues at the predicted interfaces. Based on these results, we propose that pol-beta in the open or closed conformation interacts and makes a stable interface with APEX1 bound to a cleaved abasic site on the 3' side. The method described here can be used for analysis in any DNA-metabolizing pathway where weak interactions are the principal mode of cross-talk among participants and co-crystal structures of the individual components are available.

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Subunit interacting surfaces of human haemoglobin. Localization of the alpha-subunit-beta-subunit interacting surfaces on the beta-chain by a comprehensive synthetic strategy.

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