Summary CD1 molecules function to present lipid-based antigens to T cells. Here we present the crystal structure of CD1c at 2.5 Å resolution, in complex with the pathogenic Mycobacterium tuberculosis antigen mannosyl-β1-phosphomycoketide (MPM). CD1c accommodated MPM’s methylated alkyl chain exclusively in the A′ pocket, aided by a unique exit portal underneath the α1 helix. Most striking was an open F′ pocket architecture lacking the closed cavity structure of other CD1 molecules, reminiscent of peptide binding grooves of classical Major Histocompatibility Complex molecules. This feature, combined with tryptophan-fluorescence quenching during loading of a dodecameric lipopeptide antigen, provides a compelling model by which both the lipid and peptide moieties of the lipopeptide are involved in CD1c presentation of lipopeptides.

The loss of conformational entropy is a major contribution in the thermodynamics of protein folding. However, accurate determination of the quantity has proven challenging. We calculate this loss using molecular dynamic simulations of both the native protein and a realistic denatured state ensemble. For ubiquitin, the total change in entropy is TΔS Total = 1.4 kcal·mol −1 per residue at 300 K with only 20% from the loss of side-chain entropy. Our analysis exhibits mixed agreement with prior studies because of the use of more accurate ensembles and contributions from correlated motions. Buried side chains lose only a factor of 1.4 in the number of conformations available per rotamer upon folding (Ω U /Ω N ). The entropy loss for helical and sheet residues differs due to the smaller motions of helical residues (TΔS helix−sheet = 0.5 kcal·mol NMR order parameters | molecular dynamics | helix propensity | sheet propensity | denatured state A n accurate determination of the loss of conformational entropy is critical for dissecting the energetics of reactions involving protein motions, including folding, conformational change, and binding (1-6). Given the difficulty of directly measuring the conformational entropy, most early estimates relied on computational approaches (2, 7-10), although, more recently, NMR methods have been used to measure site-resolved entropies (11). The computational methods often calculated the entropy of either the native state ensemble (NSE) or the denatured state ensemble (DSE) and invoked assumptions about the entropy of the other ensemble [e.g., assuming the NSE is a single state or that the DSE is a composite of all side-chain (SC) rotameric states in the Protein Data Bank (PDB)]. Most previous approaches focused on helices and omitted contributions from vibrations and correlated motions (12, 13), thereby partly accounting for the spectrum of calculated values.We address these issues by calculating the chain's conformational entropy from the distributions of the backbone (BB) (ϕ,ψ) and SC rotametric angles, [χ n ], obtained from all-atom simulations of the NSE and DSE for mammalian ubiquitin (Ub). This study extends our previous calculation of the loss of BB entropy that used an experimentally validated DSE (14). The calculated angular distributions reflect both the Ramachandran (Rama) basin populations and the torsional vibrations. Correlated motions are accounted for through the use of joint probability distributions [e.g., P(ϕ,ψ,χ 1 ,χ 2 )].The computed loss of BB entropy is 80% of the total entropy loss at 300 K. The BB entropy is independent of burial and residue type (excluding Pro, Gly, and pre-Pro residues) but depends on the secondary structure. Helical residues lose more BB entropy than sheet residues, TΔS helix−sheet = 0.5 kcal·mol −1 at 300 K, a difference not fully reflected by either amide N-H or carbonyl C=O bond NMR order parameters. The SC entropy loss, TΔS SC ∼ 0.2 kcal·mol −1 ·rotamer −1 , is largely independent of 2°structure and weakly correlated with burial. Combinin...

The neuronal α4β2 nicotinic acetylcholine receptor (nAChR) is one of the most widely expressed nAChR subtypes in the brain. Its subunits have high sequence identity (54% and 46% for α4 and β2, respectively) with α and β subunits in Torpedo nAChR. Using known structure of the Torpedo nAChR as a template, the closed-channel structure of the α4β2 nAChR was constructed through homology modeling. Normal mode analysis was performed on this closed structure and the resulting lowest frequency mode was applied to it for a 'twist-to-open' motion, which increased the minimum pore radius from 2.7Å to 3.4Å and generated an open-channel model. Nicotine could bind to the predicted agonist binding sites in the open-channel model, but not in the closed one. Both models were subsequently equilibrated in a ternary lipid mixture via extensive molecular dynamics (MD) simulations. Over the course of 11-ns MD simulations, the open channel remained open with filled water, but the closed channel showed a much lower water density at its hydrophobic gate comprising of residues α4-V259, α4-L263 and their homologous residues in β2 subunits. Brownian Dynamics simulations of Na + permeation through the open channel demonstrated a current-voltage relationship that was in good agreement with experimental data on the conducting state of α4β2 nAChR. Besides establishment of the well-equilibrated closed-and open-channel α4β2 structural models, the MD simulations on these models provided valuable insights into critical factors that potentially modulate channel gating. Rotation and titling of TM2 helices led to changes in orientations of pore-lining residue side-chains. Without concerted movement, the reorientation of one or two hydrophobic sidechains could be enough for channel opening. The closed-and open-channel structures exhibited distinct patterns of electrostatic interactions at the interface of extracellular and transmembrane domains that might regulate the signal propagation of agonist binding to channel opening. A potential prominent role of the β2 subunit in channel gating was also elucidated in the study.

The actin regulatory protein cofilin plays a central role in actin assembly dynamics by severing filaments and increasing the concentration of ends from which subunits add and dissociate. Cofilin binding modifies the average structure and mechanical properties of actin filaments, thereby promoting fragmentation of partially decorated filaments at boundaries of bare and cofilin-decorated segments. Despite extensive evidence for cofilin-dependent changes in filament structure and mechanics, it is unclear how the two processes are linked at the molecular level. Here, we use molecular dynamics simulations and coarse-grained analyses to evaluate the molecular origins of the changes in filament compliance due to cofilin binding. Filament subunits with bound cofilin are less flat and maintain a significantly more open nucleotide cleft than bare filament subunits. Decorated filament segments are less twisted, thinner (considering only actin), and less connected than their bare counterparts, which lowers the filament bending persistence length and torsional stiffness. Using coarse-graining as an analysis method reveals that cofilin binding increases the average distance between the adjacent long-axis filament subunit, thereby weakening their interaction. In contrast, a fraction of lateral filament subunit contacts are closer and presumably stronger with cofilin binding. A cofilactin interface contact identified by cryo-electron microscopy is unstable during simulations carried out at 310K, suggesting that this particular interaction may be short-lived at ambient temperatures. These results reveal the molecular origins of cofilin-dependent changes in actin filament mechanics that may promote filament severing.

The loss of conformational entropy is the largest unfavorable quantity affecting a protein’s stability. We calculate the reduction in the number of backbone conformations upon folding using the distribution of backbone dihedral angles (φ,ψ) obtained from an experimentally validated denatured state model, along with all-atom simulations for both the denatured and native states. The average loss of entropy per residue is TΔSBBU-N = 0.7, 0.9, or 1.1 kcal·mol−1 at T = 298 K, depending on the force field used, with a 0.6 kcal·mol−1 dispersion across the sequence. The average equates to a decrease of a factor of 3–7 in the number of conformations available per residue (f = ΩDenatured/ΩNative) or to a total of ftot=3n–7n for an n residue protein. Our value is smaller than most previous estimates where f = 7–20, i.e., our computed TΔSBBU-N is smaller by 10–100 kcal mol−1 for n=100. The differences emerge from our use of realistic native and denatured state ensembles as well as from the inclusion of accurate local sequence preferences, neighbor effects, and correlated motions (vibrations), in contrast to some previous studies that invoke gross assumptions about the entropy in either or both states. We find that the loss of entropy primarily depends on the local environment and less on properties of the native state, with the exception of α-helical residues in some force fields.