Sequence–function relationships in folding upon binding

Abstract

Folding coupled to binding is ubiquitous in biology. Nevertheless, the relationship of sequence to function for protein segments that undergo coupled binding and folding remains to be determined. Specifically, it is not known if the well-established rules that govern protein folding and stability are relevant to ligand-linked folding transitions. Upon small ligand biotinoyl-5 0 -AMP (bio-5 0 -AMP) binding the Escherichia coli protein BirA undergoes a disorder-to-order transition that results in formation of a network of packed hydrophobic side chains. Ligand binding is also allosterically coupled to protein association, with bio-5 0 -AMP binding enhancing the dimerization free energy by 24.0 kcal/mol. Previous studies indicated that single alanine replacements in a three residue hydrophobic cluster that contributes to the larger network disrupt cluster formation, ligand binding, and allosteric activation of protein association. In this work, combined equilibrium and kinetic measurements of BirA variants with alanine substitutions in the entire hydrophobic network reveal large functional perturbations resulting from any single substitution and highly non-additive effects of multiple substitutions. These substitutions also disrupt ligand-linked folding. The combined results suggest that, analogous to protein folding, functional disorder-to-order linked to binding requires optimal packing of the relevant hydrophobic side chains that contribute to the transition. The potential for many combinations of residues to satisfy this requirement implies that, although functionally important, segments of homologous proteins that undergo folding linked to binding can exhibit sequence divergence.

Heat Capacity Changes and Disorder-to-Order Transitions in Allosteric Activation

“…Previous studies indicate that any perturbation of ABL folding, either through direct alanine substitution of loop residues or through the G142A substitution, is accompanied by a 3.0−4.0 kcal/mol penalty to bio-5′-AMP binding. 13,15,18 Consequently, the absence of effects of alanine substitutions on the bio-5′-AMP binding free energy at 20°C for the remaining dimerization surface variants provides strong evidence of the preservation of the ABL disorder-to-order transition in these proteins. Structural data indicate that the G142A substitution is also accompanied by disruption of a coilto-helix transition in residues 143−146 that occurs in wtBirA ( Figure 1).…”

“…8,16 Effector binding is accompanied by folding of both the BBL and ABL with concomitant formation of a network of interacting hydrophobic side chains, and alanine substitutions in this network perturb loop folding as well as bio-5′-AMP synthesis and binding. 13,15,17 These alanine substitutions also result in changes to the coupling free energy between binding and dimerization that range from 0.5 to 1.5 kcal/mol. 13,15 Thus, the disorder-to-order transition at the effector binding site contributes to allosteric activation of dimerization.…”

“…13,15,17 These alanine substitutions also result in changes to the coupling free energy between binding and dimerization that range from 0.5 to 1.5 kcal/mol. 13,15 Thus, the disorder-to-order transition at the effector binding site contributes to allosteric activation of dimerization.…”

What Mutagenesis Can and Cannot Reveal About Allostery

“…The use of mutations becomes more challenging as extended regions of a protein (the ''great-in-between'') contribute to the function being studied and the number of positions to be probed increases. Fortunately, improved high-throughput techniques have facilitated mutagenesis studies involving large sets of mutant proteins, including mutational evaluations of allosteric mechanisms (4,17,57,(67)(68)(69)(70)(71)(72)(73)(74)(75)(76)(77)(78). A whole-protein mutagenesis design (possibly initiated with an alanine-scan) seems like a logical next step to identify residues/positions that contribute to allosteric mechanisms, and of course, a range of residues should be substituted at each position of interest.…”

Mechanisms of biotin-regulated gene expression in microbes

“… 41 When dimerization occurs, an extended intermolecular β-sheet is formed involving residues 189–195 in the central domain. 49 Following the structural changes induced by biotin, the biotin-binding loop encases the co-repressor and is stabilized through a network of hydrophobic interactions 50 as well as direct hydrogen bonding interactions involving R118. 49 Binding of the co-repressor leads to the ordering of the ATP-binding loop (residues 212–223) and better packing of the biotin-binding loop, which supports bonding interactions that stabilizes the dimer.…”