Proline Substitution of Dimer Interface β-strand Residues as a Strategy for the Design of Functional Monomeric Proteins

Abstract

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline's unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins.

Solution NMR characterization of WTCXCL8 monomer and dimer binding to CXCR1 N‐terminal domain

“…More recently, we designed the V27P/E29P mutant on the basis that disrupting backbone H‐bonding interactions across the dimer interface should result in a monomer. We have shown that the V27P/E29P mutant is indeed a monomer (referred to as the V27P/E29P monomer) and that it has WT monomer‐like activity …”

“…). The structure of a trapped monomer designed by introducing a methyl group substitution of a backbone amide proton of the dimer interface residue Leu25 is known, and is observed to be similar to the monomer of the dimer 12 ; however, amide exchange and backbone dynamics measurements have shown that the monomer is conformationally more flexible . The CXCR1 structure and solution studies of the peptides have shown that the N‐terminal domain lacks structure and is natively unfolded, but adopts a definite structure on binding …”

“…We have shown that the V27P/E29P mutant is indeed a monomer (referred to as the V27P/E29P monomer) and that it has WT monomer-like activity. 35 The binding affinities of both the 1-66 and V27P/E29P monomers for the h29mer from our NMR titrations were determined to be <5 mM [Table II, Fig. 6(B)].…”

“…The structure of a trapped monomer designed by introducing a methyl group substitution of a backbone amide proton of the dimer interface residue Leu25 is known, and is observed to be similar to the monomer of the dimer 12 ; however, amide exchange and backbone dynamics measurements have shown that the monomer is conformationally more flexible. 12,13,35 The CXCR1 structure and solution studies of the peptides have shown that the N-terminal domain lacks structure and is natively unfolded, but adopts a definite structure on binding. 26,[44][45][46] Comparison of the chemical shift perturbation profiles between the WT dimer and the V27P/E29P monomer also show that the extent of perturbation is lower for the dimer; furthermore, residues such as D52, E55, I39, V58 are highly perturbed in the monomer but less in the dimer (Fig.…”

Structure of monomeric Interleukin-8 and its interactions with the N-terminal Binding Site-I of CXCR1 by solution NMR spectroscopy

“…Non-dissociating IL-8 dimers result from the introduction of a disulfide bond across the dimerization interface (Rajarathnam et al 2006). IL-8 monomers have been produced in several ways; by replacing Leu25 at the dimerization interface with the unnatural amino acid N-methyl leucine, by mutating residues Leu25, Val27 and Glu29 to disrupt the hydrogen bonding network that stabilizes the dimeric form (Lowman et al 1997; Joseph et al 2013), and by removing the last six residues of the protein to disrupt the dimerization interface (Clark-Lewis et al 1991; Rajarathnam et al 1997), which is the monomeric form used here. The structures of each subunit of dimeric wild type IL-8 and of the monomeric IL-8 containing N -methyl leucine are similar, with some differences in the position of the first β-strand and the helical structure of residues 67–72 (Clore et al 1990; Rajarathnam et al 1995).…”