Functional modulation of a protein folding landscape via side-chain distortion

Abstract

Ultrahigh-resolution (<1.0 Å) structures have revealed unprecedented and unexpected details of molecular geometry, such as the deformation of aromatic rings from planarity. However, the functional utility of such energetically costly strain is unknown. The 0.83 Å structure of α-lytic protease (αLP) indicated that residues surrounding a conserved Phe side-chain dictate a rotamer which results in a ∼6°distortion along the side-chain, estimated to cost 4 kcal∕mol. By contrast, in the closely related protease Streptomyces griseus Protease B (SGPB), the equivalent Phe adopts a different rotamer and is undistorted. Here, we report that the αLP Phe side-chain distortion is both functional and conserved in proteases with large pro regions. Sequence analysis of the αLP serine protease family reveals a bifurcation separating those sequences expected to induce distortion and those that would not, which correlates with the extent of kinetic stability. Structural and folding kinetics analyses of family members suggest that distortion of this side-chain plays a role in increasing kinetic stability within the αLP family members that use a large Pro region. Additionally, structural and kinetic folding studies of mutants demonstrate that strain alters the folding free energy landscape by destabilizing the transition state (TS) relative to the native state (N). Although side-chain distortion comes at a cost of foldability, it suppresses the rate of unfolding, thereby enhancing kinetic stability and increasing protein longevity under harsh extracellular conditions. This ability of a structural distortion to enhance function is unlikely to be unique to αLP family members and may be relevant in other proteins exhibiting side-chain distortions.protein distortion | protein structure | X-ray crystallography | protein stability | protein lifetime T he physicochemical forces that determine macromolecular structure and function are generally thought to be well understood. The predominant forces include well-studied hydrogen bonding, van der Waals, and ionic interactions. By contrast, bonded terms are largely ignored as it is commonly held that bond lengths and angles are at or very close to canonical values in the native state. This view is so widely adopted that nearly all crystallographic and NMR structure determination methods include restraints to optimal values. However, distortion of covalent bonds in protein sidechains away from their low energy configurations has now been noted in several ultrahigh-resolution structures (1, 2) (Fig. 1A), but no functional role for the distortion has been ascribed. We examine the functional consequences of side-chain distortion in α-lytic protease (αLP), in which a 0.83 Å structure (2) revealed a surprisingly large distortion of the aromatic ring of a conserved Phe (Phe228) by almost 6°from planarity ( Fig. 1B; about 2.6 standard deviations from the mean in Fig. 1A).The αLP sub-family of serine proteases is one of the best-studied examples of a kinetically stable protein (3, 4); i.e. a protein that...

Keep on Moving: Discovering and Perturbing the Conformational Dynamics of Enzymes

“…The benzoyl moiety of Phe228 deviates by 6°from planarity. Removing this distortion can change the unfolding barrier from over a year to less than 2 weeks 73. Electron density contoured to 4.75 e − /Å 3 (dark blue mesh, lower) and 0.5 e − /Å 3 (cyan volume representation).…”

Exploring the Evolutionary History of Kinetic Stability in the α-Lytic Protease Family

“…Strain caused by the out-of-plane distortion of Phe228 in αLP and other ProL homologs has been shown to correlate with increased kinetic stability. 35 Covarying residues (I162, K165, A130, T181, W199, and Q210) 34,36 create a tightly packed arrangement around Phe228 and are important for maintaining the out-of-plane strain in αLP. Our multiple-sequence alignment together with the N4 structure confirms that Phe228 is not present in N4 and is replaced by a leucine (Figure S1 and Figure 4A).…”

“…Several unique aspects of the αLP sequence have been shown to contribute to its increased kinetic barrier. Strain caused by the out-of-plane distortion of Phe228 in αLP and other ProL homologs has been shown to correlate with increased kinetic stability . Co-varying residues (I162, K165, A130, T181, W199, and Q210) , create a tightly packed arrangement around Phe228 and are important for maintaining the out-of-plane strain in αLP.…”

Conserved Prosegment Residues Stabilize a Late-Stage Folding Transition State of Pepsin Independently of Ground States

“…Similarly, mutations that stabilize the native state yet destabilize the TS due to differences in conformational strain also produce negative Φ-values [45] . Conformational strain present in the TS but not in the native state can also give rise to Φ-values >1 [46] . In PS-pepsin the V4A mutation was stabilizing to the native state and destabilizing to the TS, suggestive of a slightly frustrated or overly-packed native state and a more optimally packed TS.…”