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'-AMP (bio-5'-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'-AMP binding enhancing the dimerization free energy by -4.0 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.

Keywords: folding upon binding; hydrophobic packing; isothermal titration calorimetry; kinetics; sedimentation equilibrium.

Figure 1

The E. coli biotin regulatory system illustrating the multiple functions of BirA (see text). Protein segments including the BBL (blue), residues 116–128, and the ABL (red), residues 210–234, are disordered (dashed line) in apoBirA and ordered in holoBirA., The boxed figure illustrates the hydrophobic network that assembles upon bio-5′-AMP binding. Color scheme: ABL cluster: V214 (light green), V219 (blue), W223 (red); ABL-BBL bridge: F124 (orange), P126 (dark green), M211 (magenta), V218 (cyan); the charged and polar side chains, which project away from the protein surface, are shown as black sticks. The models were generated using Pymol with pdb files 1BIA and 2EWN as input.

Figure 2

Sequence divergence for hydrophobic residues that assemble around the adenylate ligand in biotin ligase homologs. A. Alignment of the BBL and ABL segments of the Escherichia coli, Mycobacterium tuberculosis, and Staphylococcus aureus biotin protein ligases. The numbering system is from the E. coli protein. Color code: polar or charged residues (black), hydrophobic residues (gray), hydrophobic residues that assemble around the adenylate ligand (colored). B. Hydrophobic packing around the adenylate ligand of the I. E. coli (2EWN) II. S.a. (3V8L) and M.tb (4OP0) biotin protein ligases. The color code for the amino acids is identical to that used in A. Polar and charged side chains are shown as black sticks and the adenylate ligand as colored sticks. The alignments were extracted from alignments of the entire sequences of the three proteins using ClustalW, and the protein structure figures were prepared using Pymol.

Figure 3

Alanine substitutions result in large, non-additive effects on bio-5′-AMP binding. A. Top panel: ITC trace for V218A BirA with bio-5′-AMP: 19 13 μL volumes of 20 μM bio-5′-AMP were injected into 2 μM V218A. Bottom panel: nonlinear regression of the binding isotherm obtained from A using a single site binding model. B. Perturbations to the Gibbs free energy of bio-5′-AMP binding to the alanine substituted BirA variants shown as dark gray bars with the expected additive effects of the multiple substitutions shown as the sum of the dark gray plus light gray bars. C. Enthalpic (ΔH°) and entropic (−TΔS°) contributions to bio-5′-AMP binding for the BirA variants. The error bars were obtained from standard propagation of errors associated with at least two independent measurements.

Figure 4

Ligand-linked folding is perturbed for the majority of the BirA variants. A. Time courses of subtilisin-catalyzed cleavage of P126A BirA measured in the absence (▪) and presence (Δ) of saturating bio-5′-AMP. The lines represent the best-fit of the data to a single exponential model. B. Ratio of the pseudo first-order rates of subtilisin-mediated cleavage in the absence, k_apo, and presence, k_holo, of saturating bio-5′-AMP.

Figure 5

Alanine substituted variants are defective in catalyzing bio-5′-AMP synthesis. Kinetic transients for bio-5′-AMP synthesis by wild type ((○) and F124AV218A BirA ((Δ) with the lines representing the best-fits of the data to a single exponential equation and the residuals of the fits provided in the bottom panels.

Figure 6

Effects of alanine substitutions on ligand-linked dimerization are modest but non-additive. A. Absorbance versus radius profiles for bio-5′-AMP bound M211A variant prepared at 40, 50, and 60 μM and centrifuged at 21,000 (□) and 24,000 (○) rpm along with the best-fit curves obtained from the global analysis of nine datasets to a monomer–dimer model using WinNonLin. The lower panel shows the residuals of the fits for each of the six datasets that are shown. B. Energetic penalties to holoBirA dimerization indicated as dark gray bars with anticipated additive effects of multiple alanine substitutions provided as the sum of the dark and light gray bars.