Conserved Residues and Their Role in the Structure, Function, and Stability of Acyl-Coenzyme A Binding Protein

Abstract

In the family of acyl-coenzyme A binding proteins, a subset of 26 sequence sites are identical in all eukaryotes and conserved throughout evolution of the eukaryotic kingdoms. In the context of the bovine protein, the importance of these 26 sequence positions for structure, function, stability, and folding has been analyzed using single-site mutations. A total of 28 mutant proteins were analyzed which covered 17 conserved sequence positions and three nonconserved positions. As a first step, the influence of the mutations on the protein folding reaction has been probed, revealing a folding nucleus of eight hydrophobic residues formed between the N- and C-terminal helices [Kragelund, B. B., et al. (1999) Nat. Struct. Biol. (In press)]. To fully analyze the role of the conserved residues, the function and the stability have been measured for the same set of mutant proteins. Effects on function were measured by the extent of binding of the ligand dodecanoyl-CoA using isothermal titration calorimetry, and effects on protein stability were measured with chemical denaturation followed by intrinsic tryptophan and tyrosine fluorescence. The sequence sites that have been conserved for direct functional purposes have been identified. These are Phe5, Tyr28, Tyr31, Lys32, Lys54, and Tyr73. Binding site residues are mainly polar or charged residues, and together, four of these contribute approximately 8 kcal mol-1 of the total free energy of binding of 11 kcal mol-1. The sequence sites conserved for stability of the structure have likewise been identified and are Phe5, Ala9, Val12, Leu15, Leu25, Tyr28, Lys32, Gln33, Tyr73, Val77, and Leu80. Essentially, all of the conserved residues that maintain the stability are hydrophobic residues at the interface of the helices. Only one conserved polar residue, Gln33, is involved in stability. The results indicate that conservation of residues in homologous proteins may result from a summed optimization of an effective folding reaction, a stable native protein, and a fully active binding site. This is important in protein design strategies, where optimization of one of these parameters, typically function or stability, may influence any of the others markedly.

Identification and Characterization of a Novel Golgi Protein, GCP60, That Interacts with the Integral Membrane Protein Giantin

“…In addition, proteinprotein interactions via the proline-rich motif were reported in a vesicular transport system with dynamin (55) and in cytoskeletal architecture with ␤III spectrin (56). The second characteristic domain is an ACB domain that has a similar amino acid sequence to the lipid-binding domain of cytoplasmic acylCoA-binding proteins (35,37). In a cell-free vesicular transport assay, acyl-CoA esters including palmitoyl-CoA were shown to be essential for either the budding or the fusion process (57,58).…”

“…However, a direct target molecule for acyl-CoA involved in the process has not been identified. Although two essential tyrosine residues in the lipid-binding motif (35) are substituted with valine and phenylalanine in the ACB domain of GCP60, it is of interest to examine whether GCP60 is able to bind acyl- CoA, and whether acyl-CoA is required for the formation of the functional complex between GCP60 and giantin.…”

Binding site differences revealed by crystal structures of Plasmodium falciparum and bovine acyl-CoA binding protein

“…Tyr31, Gln33, and Lys54) play key roles in the function, stability, and folding of ACBP. 7,8 The three-dimensional structure of recombinant bovine ACBP and of ACBP complexed with palmitoyl-CoA has been determined by NMR. 9,10 It consists of four a-helices arranged in a bowl shape with a highly exposed acyl-CoA-binding site ( Figure 2).…”

Clorocatecol 1,2-dioxigenase e Proteína Ligante de Acil-CoA: caracterização estrutural e interações com ligantes

“…As diferenças mais pronunciadas acontecem à margem das interfaces e, provavelmente, se devem à maior dinâmica estrutural [51]. Em se tratando de uma proteína de um único domínio com enovelamento reversível e transição de dois estados de forma cooperativa, é frequentemente utilizada como sistema modelo para estudo de dinâmica de proteína [50,[52][53][54][55]. Mutações em resíduos conservados demonstraram que a interface hélice A1-A4 tem grande importância no processo de enovelamento da proteína.…”

“…A diferença de energia livre entre os estados desnaturado e nativo calculada para a ACBP bovina foi de ∆ − = 7,8 ± 0,2 kcal.mol -1 [54], e os resíduos que contribuem com o enovelamento são responsáveis pela formação de interações hidrofóbicas entre as hélices A1, A2 e A4 e também entre A2 e A3 [54]. Por outro lado, a mutação de resíduos carregados não desestabiliza a estrutura significativamente [54].…”