Betaalpha-hairpin clamps brace betaalphabeta modules and can make substantive contributions to the stability of TIM barrel proteins

Abstract

Non-local hydrogen bonding interactions between main chain amide hydrogen atoms and polar side chain acceptors that bracket consecutive betaalpha or alphabeta elements of secondary structure in alphaTS from E. coli, a TIM barrel protein, have previously been found to contribute 4-6 kcal mol(-1) to the stability of the native conformation. Experimental analysis of similar betaalpha-hairpin clamps in a homologous pair of TIM barrel proteins of low sequence identity, IGPS from S. solfataricus and E. coli, reveals that this dramatic enhancement of stability is not unique to alphaTS. A survey of 71 TIM barrel proteins demonstrates a 4-fold symmetry for the placement of betaalpha-hairpin clamps, bracing the fundamental betaalphabeta building block and defining its register in the (betaalpha)(8) motif. The preferred sequences and locations of betaalpha-hairpin clamps will enhance structure prediction algorithms and provide a strategy for engineering stability in TIM barrel proteins.

Figure 1. Ribbon diagrams of sIGPS (A) and eIGPS (B) highlighting the βα-hairpin clamps.

(C) and (D) display the intervening elements of secondary structures between the residues forming the clamps for sIGPS: sIGPS-β2α2-S104_NH→_Oε1E74; sIGPS-β3α3-I107_NH→_Oδ1D128; and sIGPS-β7α7-K207_NH→_Oδ2N228 and for eIGPS: eIGPS-β1α1-F50_NH→_OγS82; eIGPS-β3α3-I111_NH→_Oδ2D132; and eIGPS-β7α7-V211_NH→_Oδ1N231. The SCs involved in the clamp interactions are highlighted with the H-bond donor and acceptor atoms shown in blue and red, respectively. The distances between the donor and acceptor atoms are indicated. The solvent exposed surface areas of the H-bond donor and acceptor atoms is shown in parenthesis. The H-bonds and their corresponding distances were determined by using the program HBPLUS . The structures were generated using PyMOL v 0.99 , and the PDB codes are 2C3Z for sIGPS and 1PII for eIGPS .

Figure 2. Ellipticity of wild-type and clamp-deletion variants of sIGPS and eIGPS.

Far-UV (a, b) and near-UV (c, d) CD spectra of sIGPS (a) and (c): sIGPS-WT (–––––), sIGPS-Δβ2α2-E74A (– • –), sIGPS-Δβ3α3-D128A (•••), and sIGPS-Δβ7α7-N228A (– –); and eIGPS (b) and (d): eIGPS-WT (–––––), eIGPS-Δβ1α1-S82A (– • –), eIGPS-Δβ3α3-D132A (•••), and eIGPS-Δβ7α7-N231A (– –). Buffer conditions: 10 mM potassium phosphate, 0.2 mM K₂EDTA, 1 mM βME, pH 7.8 for sIPGS and pH 7.0 for eIGPS at 25°C.

Figure 3. Stability perturbation of sIGPS and eIGPS by clamp deletion.

(A) and (B) display urea denaturation equilibrium unfolding curves of WT and clamp-deletion variants of IGPS, the lines represent fits of the data for each variant to a 3-state equilibrium folding model as described in the text. (a) sIGPS: sIGPS-WT (•,–––––), sIGPS-Δβ2α2-E74A (▴,– • –), sIGPS-Δβ3α3-D128A (⧫,•••), and sIGPS-Δβ7α7-N228A (▪,– –). (b) eIGPS: eIGPS-WT (•,–––––), eIGPS-Δβ1α1-S82A (▴,– • –), eIGPS-Δβ3α3-D132A (⧫,•••), and eIGPS-Δβ7α7-N231A (▪,– –). (C) and (D) are bar graphs representing the free energy differences for the N to I step in unfolding, ΔG°_NI, (black bars) and the I to U step, ΔG°_IU (gray bars) for WT and the clamp-deletion variants of sIGPS (C) and eIGPS (D). The urea denaturation equilibrium unfolding curve of sIGPS-WT (A) and the corresponding folding free energy changes (C) are adapted from Forsyth et al. .

Figure 4. Positional preference of βα-hairpin clamps in 71 TIM barrel proteins.

(A) The TIM barrel architecture is represented by a cross-sectional view of the 8 β-strands, represented as rectangles and the strand number is indicated. The number of MC_NH → SC βα-hairpin clamp interactions connecting adjacent β-strands with SC H-bond acceptors C-terminal to the MC_NH donors (–––––), and with SC H-bond acceptors N-terminal to the MC_NH donors (– –) are indicated. The number of βα-hairpin clamps with (I/L/V) MC → SC (D) is represented in parenthesis. (B) The positional preference of (I/L/V) MC → SC (D) relative to the β-strands. The MC donor prefers either the first or second position of the β-strand and the SC acceptor prefers to be in the loop immediately preceding the subsequent β-strand. The number of times each pair of interactions occurs in the 55 I/L/V MC → SC D sub-set is indicated.

Figure 5. Architectural principles of the TIM barrel fold.

The strand number of the 8 β-strands of the TIM barrel architecture (↗) is indicated at the C-terminus of each β-strand. To convey the closed barrel architecture, β8 is shown adjacent to β1 (↗) as well as adjacent to β7. The position of each residue on the β-strands, with SC pointing into the β-barrel (••) and SC pointing towards the α-helices (), is indicated. The one letter code for the most common amino acids (>15%) in the loop preceding the β-strand in the 71 TIM barrel proteins database is shown. The H-bond network for the β-barrel (), the βα-hairpin clamp interactions between the second residue of an odd-numbered β-strand and the side chain of the residue immediately preceding the subsequent even-numbered β-strand (→), and the MC-MC interactions between the same two residues (− →) are indicated.