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Tissue folds are structural motifs critical to organ function. In the intestine, bending of a flat epithelium into a periodic pattern of folds gives rise to villi, finger-like protrusions that enable nutrient absorption. However, the molecular and mechanical processes driving villus morphogenesis remain unclear. Here, we identify an active mechanical mechanism that simultaneously patterns and folds the intestinal epithelium to initiate villus formation. At the cellular level, we find that PDGFRA+ subepithelial mesenchymal cells generate myosin II-dependent forces sufficient to produce patterned curvature in neighboring tissue interfaces. This symmetry-breaking process requires altered cell and extracellular matrix interactions that are enabled by matrix metalloproteinase-mediated tissue fluidization. Computational models, together with in vitro and in vivo experiments, revealed that these cellular features manifest at the tissue level as differences in interfacial tensions that promote mesenchymal aggregation and interface bending through a process analogous to the active dewetting of a thin liquid film., Competing Interests: Declaration of interests Z.J.G. and C.S.M. hold patents related to the MULTI-seq barcoding method., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The continual emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) poses a major challenge to vaccines and antiviral therapeutics due to their extensive evasion of immunity. Aiming to develop potent and broad-spectrum anticoronavirus inhibitors, we generated A1-(GGGGS)7-HR2m (A1L35HR2m) by introducing an angiotensin-converting enzyme 2 (ACE2)-derived peptide A1 to the N terminus of the viral HR2-derived peptide HR2m through a long flexible linker, which showed significantly improved antiviral activity. Further cholesterol (Chol) modification at the C terminus of A1L35HR2m greatly enhanced the inhibitory activities against SARS-CoV-2, SARS-CoV-2 VOCs, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) pseudoviruses, with IC 50 values ranging from 0.16 to 5.53 nM. A1L35HR2m-Chol also potently inhibits spike-protein-mediated cell-cell fusion and the replication of authentic Omicron BA.2.12.1, BA.5, and EG.5.1. Importantly, A1L35HR2m-Chol distributed widely in respiratory tract tissue and had a long half-life (>10 h) in vivo. Intranasal administration of A1L35HR2m-Chol to K18-hACE2 transgenic mice potently inhibited Omicron BA.5 and EG.5.1 infection both prophylactically and therapeutically., Competing Interests: Declaration of interests B.D., W.B., S.Y., K.T., and G.C. are the inventors of a provisional patent filed by Westlake University and The University of Hong Kong., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Deregulated HER2 is a target of many approved cancer drugs. We analyzed 111,176 patient tumors and identified recurrent mutations in HER2 transmembrane domain (TMD) and juxtamembrane domain (JMD) that include G660D, R678Q, E693K, and Q709L. Using a saturation mutagenesis screen and testing of patient-derived mutations we found several activating TMD and JMD mutations. Structural modeling and analysis showed that the TMD/JMD mutations function by improving the active dimer interface or stabilizing an activating conformation. Further, we found that HER2 G660D employed asymmetric kinase dimerization for activation and signaling. Importantly, anti-HER2 antibodies and small-molecule kinase inhibitors blocked the activity of TMD/JMD mutants. Consistent with this, a G660D germline mutant lung cancer patient showed remarkable clinical response to HER2 blockade., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We present a strategy for designed self-assembly of peptides into two-dimensional monolayer crystals on the surface of graphene and graphite. As predicted by computation, designed peptides assemble on the surface of graphene to form very long, parallel, in-register β-sheets, which we call β-tapes. Peptides extend perpendicularly to the long axis of each β-tape, defining its width, with hydrogen bonds running along the axis. Tapes align on the surface to create highly regular microdomains containing 4-nm pitch striations. Moreover, in agreement with calculations, the atomic structure of the underlying graphene dictates the arrangement of the β-tapes, as they orient along one of six directions defined by graphene's sixfold symmetry. A cationic-assembled peptide surface is shown here to strongly adhere to DNA, preferentially orienting the double helix along β-tape axes. This orientational preference is well anticipated from calculations, given the underlying peptide layer structure. These studies illustrate how designed peptides can amplify the Ångstrom-level atomic symmetry of a surface onto the micrometer scale, further imparting long-range directional order onto the next level of assembly. The remarkably stable nature of these assemblies under various environmental conditions suggests applications in enzymelike catalysis, biological interfaces for cellular recognition, and two-dimensional platforms for studying DNA-peptide interactions., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Histidine kinases (HKs) are major players in bacterial signaling. There has been an explosion of new HK crystal structures in the last 5 years. We globally analyze the structures of HKs to yield insights into the mechanisms by which signals are transmitted to and across protein structures in this family. We interpret known enzymological data in the context of new structural data to show how asymmetry across the dimer interface is a key feature of signal transduction in HKs, and discuss how different HK domains undergo asymmetric to symmetric transitions during signal transduction and catalysis. A thermodynamic framework for signaling that encompasses these various properties is presented, and the consequences of weak thermodynamic coupling are discussed. The synthesis of observations from enzymology, structural biology, protein engineering, and thermodynamics paves the way for a deeper molecular understanding of HK signal transduction., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

α Helices are a basic unit of protein secondary structure and therefore the interaction between helices is crucial to understanding tertiary and higher-order folds. Comparing subtle variations in the structural and sequence motifs between membrane and soluble proteins sheds light on the different constraints faced by each environment and elucidates the complex puzzle of membrane protein folding. Here, we demonstrate that membrane and water-soluble helix pairs share a small number of similar folds with various interhelical distances. The composition of the residues that pack at the interface between corresponding motifs shows that hydrophobic residues tend to be more enriched in the water-soluble class of structures and small residues in the transmembrane class. The latter group facilitates packing via sidechain- and backbone-mediated hydrogen bonds within the low-dielectric membrane milieu. The helix-helix interactome space, with its associated sequence preferences and accompanying hydrogen-bonding patterns, should be useful for engineering, prediction, and design of protein structure., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Bacteria transduce signals across the membrane using two-component systems (TCSs), consisting of a membrane-spanning sensor histidine kinase and a cytoplasmic response regulator. In gram-negative bacteria, the PhoPQ TCS senses cations and antimicrobial peptides, yet little is known about the structural changes involved in transmembrane signaling. We construct a model of PhoQ signal transduction using Bayesian inference, based on disulfide crosslinking data and homologous crystal structures. The data are incompatible with a single conformation but are instead consistent with two interconverting structures. These states differ in membrane depth of the periplasmic acidic patch and the reciprocal displacement of diagonal helices along the dimer interface. Studies of multiple histidine kinases suggest this repacking might be a common mode of signal transduction in sensor His-kinase receptors. Because a similar scissors model has been ruled out in CheA-linked chemoreceptors, the evidence suggests that sensor His-kinase and CheA-linked receptors possess different signaling mechanisms., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Channel gating and proton conductance of the influenza A virus M2 channel result from complex pH-dependent interactions involving the pore-lining residues His37, Trp41, and Asp44. Protons diffusing from the outside of the virus protonate His37, which opens the Trp41 gate and allows one or more protons to move into the virus interior. The Trp41 gate gives rise to a strong asymmetry in the conductance, favoring rapid proton flux only when the outside is at acid pH. Here, we show that the proton currents recorded for mutants of Asp44, including D44N found in the A/FPV/Rostock/34 strain, lose this asymmetry. Moreover, NMR and MD simulations show that the mutations induce a conformational change similar to that induced by protonation of His37 at low pH, and decrease the structural stability of the hydrophobic seal associated with the Trp41 gate. Thus, Asp44 is able to determine two important properties of the M2 proton channel., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Plasmodium falciparum pathogenesis is affected by various cell types in the blood, including platelets, which can kill intraerythrocytic malaria parasites. Platelets could mediate these antimalarial effects through human defense peptides (HDPs), which exert antimicrobial effects by permeabilizing membranes. Therefore, we screened a panel of HDPs and determined that human platelet factor 4 (hPF4) kills malaria parasites inside erythrocytes by selectively lysing the parasite digestive vacuole (DV). PF4 rapidly accumulates only within infected erythrocytes and is required for parasite killing in infected erythrocyte-platelet cocultures. To exploit this antimalarial mechanism, we tested a library of small, nonpeptidic mimics of HDPs (smHDPs) and identified compounds that kill P. falciparum by rapidly lysing the parasite DV while sparing the erythrocyte plasma membrane. Lead smHDPs also reduced parasitemia in a murine malaria model. Thus, identifying host molecules that control parasite growth can further the development of related molecules with therapeutic potential., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The complex hydrophobic and hydrophilic milieus of membrane-associated proteins pose experimental and theoretical challenges to their understanding. Here, we produce a nonredundant database to compute knowledge-based asymmetric cross-membrane potentials from the per-residue distributions of C(β), C(γ) and functional group atoms. We predict transmembrane and peripherally associated regions from genomic sequence and position peptides and protein structures relative to the bilayer (available at http://www.degradolab.org/ez). The pseudo-energy topological landscapes underscore positional stability and functional mechanisms demonstrated here for antimicrobial peptides, transmembrane proteins, and viral fusion proteins. Moreover, experimental effects of point mutations on the relative ratio changes of dual-topology proteins are quantitatively reproduced. The functional group potential and the membrane-exposed residues display the largest energetic changes enabling to detect native-like structures from decoys. Hence, focusing on the uniqueness of membrane-associated proteins and peptides, we quantitatively parameterize their cross-membrane propensity, thus facilitating structural refinement, characterization, prediction, and design., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The tuning of the pK(a) of ionizable residues plays a critical role in various protein functions, such as ligand-binding, catalysis, and allostery. Proteins harness the free energy of folding to position ionizable groups in highly specific environments that strongly affect their pK(a) values. To investigate the interplay among protein folding kinetics, thermodynamics, and pK(a) modulation, we introduced a pair of Asp residues at neighboring interior positions of a coiled-coil. A single Asp residue was replaced for an Asn side chain at the a-position of the coiled-coil from GCN4, which was also crosslinked at the C-terminus via a flexible disulfide bond. The thermodynamic and kinetic stability of the system was measured by circular dichroism and stopped-flow fluorescence as a function of pH and concentration of guanidine HCl. Both sets of data are consistent with a two-state equilibrium between fully folded and unfolded forms. Distinct pK(a) values of 6.3 and 5.35 are assigned to the first and second protonation of the Asp pair; together they represent an energetic difference of 5 kcal/mol relative to the protonation of two Asp residues with unperturbed pK(a) values. Analysis of the rate data as a function of pH and denaturant concentration allowed calculation of the kinetic constants for the conformational transitions of the peptide with the Asp residues in the doubly protonated, singly protonated, and unprotonated forms. The doubly and singly protonated forms fold rapidly, and a ϕ-value analysis shows that their contribution to folding occurs subsequent to the transition state ensemble for folding. By contrast, the doubly charged state shows a reduced rate of folding and a ϕ-value near 0.5 indicative of a repulsive interaction, and possibly also heterogeneity in the transition state ensemble., (Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

We explore the interplay between amino acid sequence, thermodynamic stability, and functional fitness in the M2 proton channel of influenza A virus. Electrophysiological measurements show that drug-resistant mutations have minimal effects on M2's specific activity, and suggest that resistance is achieved by altering a binding site within the pore rather than a less direct allosteric mechanism. In parallel, we measure the effects of these mutations on the free energy of assembling the homotetrameric transmembrane pore from monomeric helices in micelles and bilayers. Although there is no simple correlation between the evolutionary fitness of the mutants and their stability, all variants formed more stable tetramers in bilayers, and the least-fit mutants showed the smallest increase in stability upon moving from a micelle to a bilayer environment. We speculate that the folding landscape of a micelle is rougher than that of a bilayer, and more accommodating of conformational variations in nonoptimized mutants.

Single-span transmembrane (TM) helices have structural and functional roles well beyond serving as mere anchors to tether water-soluble domains in the vicinity of the membrane. They frequently direct the assembly of protein complexes and mediate signal transduction in ways analogous to small modular domains in water-soluble proteins. This review highlights different sequence and structural motifs that direct TM assembly and discusses their roles in diverse biological processes. We believe that TM interactions are potential therapeutic targets, as evidenced by natural proteins that modulate other TM interactions and recent developments in the design of TM-targeting peptides.

We report the solution NMR structure of a designed dimetal-binding protein, di-Zn(II) DFsc, along with a secondary refinement step employing molecular dynamics techniques. Calculation of the initial NMR structural ensemble by standard methods led to distortions in the metal-ligand geometries at the active site. Unrestrained molecular dynamics using a nonbonded force field for the metal shell, followed by quantum mechanical/molecular mechanical dynamics of DFsc, were used to relax local frustrations at the dimetal site that were apparent in the initial NMR structure and provide a more realistic description of the structure. The MD model is consistent with NMR restraints, and in good agreement with the structural and functional properties expected for DF proteins. This work demonstrates that NMR structures of metalloproteins can be further refined using classical and first-principles molecular dynamics methods in the presence of explicit solvent to provide otherwise unavailable insight into the geometry of the metal center.

Partially folded intermediates have been frequently observed in equilibrium and kinetic protein folding studies. However, folding intermediates that exist at the native side of the rate-limiting step are rather difficult to study because they often evade detection by conventional folding kinetic methods. Here, we demonstrated that a laser-induced temperature-jump method can potentially be used to identify the existence of such post-transition or hidden intermediates. Specifically, we studied two cross-linked variants of GCN4-p1 coiled-coil. The GCN4 leucine zipper has been studied extensively and most of these studies have regarded it as a two-state folder. Our static circular dichroism and infrared data also indicate that the thermal unfolding of these two monomeric coiled-coils can be adequately described by an apparent two-state model. However, their temperature-jump-induced relaxation kinetics exhibit non-monoexponential behavior, dependent upon sequence and temperature. Taken together, our results support a folding mechanism wherein at least one folding intermediate populates behind the main rate-limiting step.

A peptide containing glycine at a and d positions of a heptad motif was synthesized to investigate the possibility that membrane-soluble peptides with a Gly-based, left-handed helical packing motif would associate. Based on analytical ultracentrifugation in C14-betaine detergent micelles, the peptide did associate in a monomer-dimer equilibrium, although the association constant was significantly less than that reported for the right-handed dimer of the glycophorin A transmembrane peptide in similar detergents. Fluorescence resonance energy transfer (FRET) experiments conducted on peptides labeled at their N-termini with either tetramethylrhodamine (TMR) or 7-nitrobenz-2-oxa-1,3-diazole (NBD) also indicated association. However, analysis of the FRET data using the usual assumption of complete quenching for NBD-TMR pairs in the dimer could not be quantitatively reconciled with the analytical ultracentrifugation-measured dimerization constant. This led us to develop a general treatment for the association of helices to either parallel or antiparallel structures of any aggregation state. Applying this treatment to the FRET data, constraining the dimerization constant to be within experimental uncertainty of that measured by analytical ultracentrifugation, we found the data could be well described by a monomer-dimer equilibrium with only partial quenching of the dimer, suggesting that the helices are most probably antiparallel. These results also suggest that a left-handed Gly heptad repeat motif can drive membrane helix association, but the affinity is likely to be less strong than the previously reported right-handed motif described for glycophorin A.

A key element of membrane fusion reactions in biology is the involvement of specific fusion proteins. In many viruses, the proteins that mediate membrane fusion usually exist as homotrimers. Furthermore, they contain extended triple-helical coiled-coil domains and fusogenic peptides. It has been suggested that the coiled-coil domains present the fusogenic peptide in a conformation or geometry favorable for membrane fusion. To test the hypothesis that trimerization of fusogenic peptide is related to optimal fusion, we have designed and synthesized a triple-stranded coiled-coil X31 peptide, also known as the ccX31, which mimics the influenza virus hemagglutinin fusion peptide in the fusion-active state. We compared the membrane interactive properties of ccX31 versus the monomeric X31 fusogenic peptide. Our data show that trimerization enhances peptide-induced leakage of liposomal contents and lipid mixing. Furthermore, studies using micropipette aspiration of single vesicles reveal that ccX31 decreases lysis tension, tau(lysis), but not area expansion modulus, Ka, of phospholipid bilayers, whereas monomeric X31 peptide lowers both tau(lysis) and Ka. Our results are consistent with the hypothesis that oligomerization of fusogenic peptide promotes membrane fusion, possibly by enhancing localized destabilization of lipid bilayers.

Thermodynamics studies aimed at quantitatively characterizing free energy effects of amino acid substitutions are not restricted to two state systems, but do require knowing the number of states involved in the equilibrium under consideration. Using analytical ultracentrifugation and NMR methods, we show here that a membrane-soluble peptide, MS1, designed by modifying the sequence of the water-soluble coiled-coil GCN4-P1, exhibits a reversible monomer-dimer-trimer association in detergent micelles with a greater degree of cooperativity in C14-betaine than in dodecyl phosphocholine detergents.

Background: A large energy gap between the native state and the non-native folded states is required for folding into a unique three-dimensional structure. The features that define this energy gap are not well understood, but can be addressed using de novo protein design. Previously, alpha(2)D, a dimeric four-helix bundle, was designed and shown to adopt a native-like conformation. The high-resolution solution structure revealed that this protein adopted a bisecting U motif. Glu7, a solvent-exposed residue that adopts many conformations in solution, might be involved in defining the unique three-dimensional structure of alpha(2)D., Results: A variety of hydrophobic and polar residues were substituted for Glu7 and the dynamic and thermodynamic properties of the resulting proteins were characterized by analytical ultracentrifugation, circular dichroism spectroscopy, and nuclear magnetic resonance spectroscopy. The majority of substitutions at this solvent-exposed position had little affect on the ability to fold into a dimeric four-helix bundle. The ability to adopt a unique conformation, however, was profoundly modulated by the residue at this position despite the similar free energies of folding of each variant., Conclusions: Although Glu7 is not involved directly in stabilizing the native state of alpha(2)D, it is involved indirectly in specifying the observed fold by modulating the energy gap between the native state and the non-native folded states. These results provide experimental support for hypothetical models arising from lattice simulations of protein folding, and underscore the importance of polar interfacial residues in defining the native conformations of proteins.

The finite-difference Poisson-Boltzmann methodology was applied to a series of parallel, alpha-helical bundle models of the designed ion channel peptide Ac-(LSSLLSL)3-CONH2. This method is able to fully describe the current-voltage curves for this channel and quantitatively explains their cation selectivity and rectification. We examined a series of energy-minimized models representing different aggregation states, side-chain rotamers, and helical rotations, as well as an ensemble of structures from a molecular dynamics trajectory. Potential energies were computed for single, permeating K+ and Cl- ions at a series of positions along a central pathway through the models. A variable-electric-field Nernst-Planck electrodiffusion model was used, with two adjustable parameters representing the diffusion coefficients of K+ and Cl- to scale the individual ion current magnitudes. The ability of a given DelPhi potential profile to fit the experimental data depended strongly on the magnitude of the desolvation of the permeating ion. Below a pore radius of 3.8 A, the predicted profiles showed large energy barriers, and the experimental data could be fit only with unrealistically high values for the K+ and Cl- diffusion coefficients. For pore radii above 3.8 A, the desolvation energies were 2kT or less. The electrostatic calculations were sensitive to positioning of the Ser side chains, with the best fits associated with maximum exposure of the Ser side-chain hydroxyls to the pore. The backbone component was shown to be the major source of asymmetry in the DelPhi potential profiles. Only two of the energy-minimized structures were able to explain the experimental data, whereas an average of the dynamics structures gave excellent agreement with experimental results. Thus this method provides a promising approach to prediction of current-voltage curves from three-dimensional structures of ion channel proteins.

The cytotoxic peptide from honeybee venom, melittin, and a synthetic peptide analogue of it lyse human erythrocytes in a biphasic process. The kinetics of the lysis in 0.30 M sucrose, 0.01 M sodium phosphate, pH 7.30 at 4 degrees C were investigated. Our results show that melittin rapidly binds to the outer surface of the erythrocyte membrane, and the surface-bound monomers produce transient openings through which approximately 40 hemoglobin molecules can escape. Concomitantly, the melittin loses its ability to effect the process, presumably by translocation through the bilayer. The half-life for this process is 1.2 min. In a much slower process, dimers of this internalized melittin again produce transient membrane openings in a steady state. On a molar basis, the synthetic peptide analogue produces a fast process comparable to that caused by melittin, but is more efficient in the slow phase. Escape of hemoglobin and of carbonic anhydrase through the openings is diffusion controlled. These results suggest that the functional units necessary for the activity of melittin-like cytotoxic peptides are a 20 amino acid amphiphilic alpha-helix with a hydrophobic:hydrophilic ratio greater than 1 and a short segment with a high concentration of positive charges.