Molecular Switch between Structural Compaction and Thermodynamic Stability by the Xxx-Pro Interface in Transmembrane ß-Barrels.

Transmembrane ß-barrel scaffolds found in outer membrane proteins are formed and stabilized by a defined pattern of interstrand intraprotein H-bonds, in hydrophobic lipid bilayers. Introducing the conformationally constrained proline in ß-barrels can cause significant destabilization of these structural regions that require H-bonding, with proline additionally acting as a secondary structure breaker. Membrane protein ß-barrels are therefore expected to show poor tolerance to the presence of a transmembrane proline. Here, we assign a thermodynamic measure for the extent to which a single proline can be tolerated at the C-terminal interface of the model transmembrane ß-barrel PagP. We find that proline drastically destabilizes PagP by 7.0 kcal mol-1 with respect to wild-type PagP (F161 → P161). Interestingly, strategic modulation of the preceding residue can modify the measured energetics. Placing a hydrophobic or bulky side chain as the preceding residue increases the thermodynamic stability by ≤8.0 kcal mol-1. While polar substituents at the preceding residue decrease the PagP stability, these residues demonstrate stronger tertiary packing interactions in the barrel and retain the catalytic activity of native PagP. This biophysical interplay between enhanced thermodynamic stability and attaining a structurally compact functional ß-barrel scaffold highlights the detrimental effect caused by proline incorporation. Our findings also reveal alternative mechanisms that protein sequences can employ to salvage the structural integrity of transmembrane protein structures.

Hydrophobic Characteristic Is Energetically Preferred for Cysteine in a Model Membrane Protein.

The naturally occurring amino acid cysteine has often been implicated with a crucial role in maintaining protein structure and stability. An intriguing duality in the intrinsic hydrophobicity of the cysteine side chain is that it exhibits both polar as well as hydrophobic characteristics. Here, we have utilized a cysteine-scanning mutational strategy on the transmembrane ß-barrel PagP to examine the membrane depth-dependent energetic contribution of the free cysteine side chain (thiolate) versus the parent residue at an experimental pH of 9.5 in phosphatidylcholine vesicles. We find that introduction of cysteine causes destabilization at several of the 26 lipid-facing sites of PagP that we mutated in this study. The destabilization is minimal (0.5-1.5 kcal/mol) when the mutation is toward the bilayer midplane, whereas it is higher in magnitude (3.0-5.0 kcal/mol) near the bilayer interface. These observations suggest that cysteine forms more favorable interactions with the hydrophobic lipid core as compared to the amphiphilic water-lipid interface. The destabilizing effect is more pronounced when cysteine replaces the interfacial aromatics, which are known to participate in tertiary interaction networks in transmembrane ß-barrels. Our observations from experiments involving the introduction of cysteine at the bilayer midplane further strengthen previous views that the free cysteine side chain does possess strongly apolar characteristics. Additionally, the free energy changes observed upon cysteine incorporation show a depth-dependent correlation with the estimated energetic cost of partitioning derived from reported hydrophobicity scales. Our results and observations from the thermodynamic analysis of the PagP barrel may explain why cysteine, despite possessing a polar sulfhydryl group, tends to behave as a hydrophobic (rather than polar) residue in folded protein structures.

Salvaging the Thermodynamic Destabilization of Interface Histidine in Transmembrane ß-Barrels.

The ability of histidine to participate in a wide range of stabilizing polar interactions preferentially populates this residue in functionally important sites of proteins. Histidine possesses an amphiphilic and electrostatic nature that is essential for amino acids residing at membrane interfaces. However, the frequency of occurrence of histidine at membrane interfaces, particularly transmembrane ß-barrels, is lower than those of other aromatic residues. Here, we carry out comprehensive energetic measurements using equilibrium folding of the outer membrane enzyme PagP to address the contribution of a C-terminal interface histidine to barrel stability. We show that placing histidine at the C-terminus universally destabilizes PagP by 4.0-8.0 kcal mol-1 irrespective of the neighboring residue. Spectroscopic and electrophoretic measurements indicate that the altered stability may arise from a loss of barrel compaction. Isoleucine, methionine, and valine salvage this destabilization marginally (in addition to tyrosine, which shows an exceptionally high folding free energy value), when placed at the penultimate position, at the expense of an altered folding pathway. Double-mutant cycle analysis indicates that the coupling energy between the terminal and penultimate residues in PagP-X160H161 increases when the level of intrinsic destabilization by the terminal H161 is high. Our observations that neighboring residues cannot salvage the energetic destabilization of histidine may explain why histidine is less abundant at membrane interfaces.

Energetics of side-chain partitioning of ß-signal residues in unassisted folding of a transmembrane ß-barrel protein.

The free energy of water-to-interface amino acid partitioning is a major contributing factor in membrane protein folding and stability. The interface residues at the C terminus of transmembrane ß-barrels form the ß-signal motif required for assisted ß-barrel assembly in vivo but are believed to be less important for ß-barrel assembly in vitro Here, we experimentally measured the thermodynamic contribution of all 20 amino acids at the ß-signal motif to the unassisted folding of the model ß-barrel protein PagP. We obtained the partitioning free energy for all 20 amino acids at the lipid-facing interface (ΔΔG0w,i(φ)) and the protein-facing interface (ΔΔG0w,i(π)) residues and found that hydrophobic amino acids are most favorably transferred to the lipid-facing interface, whereas charged and polar groups display the highest partitioning energy. Furthermore, the change in non-polar surface area correlated directly with the partitioning free energy for the lipid-facing residue and inversely with the protein-facing residue. We also demonstrate that the interface residues of the ß-signal motif are vital for in vitro barrel assembly, because they exhibit a side chain-specific energetic contribution determined by the change in nonpolar accessible surface. We further establish that folding cooperativity and hydrophobic collapse are balanced at the membrane interface for optimal stability of the PagP ß-barrel scaffold. We conclude that the PagP C-terminal ß-signal motif influences the folding cooperativity and stability of the folded ß-barrel and that the thermodynamic contributions of the lipid- and protein-facing residues in the transmembrane protein ß-signal motif depend on the nature of the amino acid side chain.

Distinct Structural Elements Govern the Folding, Stability, and Catalysis in the Outer Membrane Enzyme PagP.

The outer membrane enzyme PagP is indispensable for lipid A palmitoylation in Gram-negative bacteria and has been implicated in resistance to host immune defenses. PagP possesses an unusual structure for an integral membrane protein, with a highly dynamic barrel domain that is tilted with respect to the membrane normal. In addition, it contains an N-terminal amphipathic helix. Recent functional and structural studies have shown that these molecular factors are critical for PagP to carry out its function in the challenging environment of the bacterial outer membrane. However, the precise contributions of the N-helix to folding and stability and residues that can influence catalytic rates remain to be addressed. Here, we identify a sequence-dependent stabilizing role for the N-terminal helix of PagP in the measured thermodynamic stability of the barrel. Using chimeric barrel sequences, we show that the Escherichia coli PagP N-terminal helix confers 2-fold greater stability to the Salmonella typhimurium barrel. Further, we find that the W78F substitution in S. typhimurium causes a nearly 20-fold increase in the specific activity in vitro for the phospholipase reaction, compared to that of E. coli PagP. Here, phenylalanine serves as a key regulator of catalysis, possibly by increasing the reaction rate. Through coevolution analysis, we detect an interaction network between seemingly unrelated segments of this membrane protein. Exchanging the structural and functional features between homologous PagP enzymes from E. coli and S. typhimurium has provided us with an understanding of the molecular factors governing PagP stability and function.

Residue-Dependent Thermodynamic Cost and Barrel Plasticity Balances Activity in the PhoPQ-Activated Enzyme PagP of Salmonella typhimurium.

PagP is an eight-stranded transmembrane ß-barrel enzyme indispensable for lipid A palmitoylation in Gram-negative bacteria. The severity of infection by pathogens, including Salmonella, Legionella, and Bordetella, and resistance to antimicrobial peptides, relies on lipid A remodeling by PagP, rendering PagP a sought-after drug target. Despite a conserved sequence, more robust palmitoylation of lipid A is observed in Salmonella typhimurium compared to Escherichia coli, a possible consequence of the differential regulation of PagP expression and/or specific activity. Work here identifies molecular signatures that demarcate thermodynamic stability and variances in catalytic efficiency between S. typhimurium (PagP-St) and E. coli (PagP-Ec) transmembrane PagP barrel variants. We demonstrate that Salmonella PagP displays a 2-fold destabilization of the barrel, while achieving 15-20 magnitude higher lipase efficiency, through subtle alterations of lipid-facing residues distal from the active site. We find that catalytic properties of these homologues are retained across different lipid environments such as micelles, vesicles, and natural extracts. By comparing thermodynamic stability with activity of selectively designed mutants, we conclude that activity-stability trade-offs can be influenced by factors secluded from the catalytic region. Our results provide a compelling correlation of the primary protein structure with enzymatic activity, barrel thermodynamic stability, and scaffold plasticity. Our analysis can open avenues for the development of potent pharmaceuticals against salmonellosis.