Residue-specific Interactions Research Articles

Key charged residues influence the amyloidogenic propensity of the helix-1 region of serum amyloid A

Increased plasma levels of serum amyloid A (SAA), an acute-phase protein that is secreted in response to inflammation, may lead to the accumulation of amyloid in various organs thereby obstructing their functions. Severe cases can lead to a systemic disorder called AA amyloidosis. Previous studies suggest that the N-terminal helix is the most amyloidogenic region of SAA. Moreover, computational studies implicated a significant role for Arg-1 and the residue-specific interactions formed during the fibrillization process. With a focus on the N-terminal region of helix-1, SAA1–13, mutational analysis was employed to interrogate the roles of the amino acid residues, Arg-1, Ser-5, Glu-9, and Asp-12. The truncated SAA1–13 fragment was systematically modified by substituting the key residues with alanine or uncharged but structurally similar amino acids. We monitored the changes in the amyloidogenic propensities, associated conformational markers, and morphology of the amyloids resulting from the mutation of SAA1–13. Mutating out Arg-1 resulted in much reduced aggregation propensity and a lack of detectable β-structures alluding to the importance of salt-bridge interactions involving Arg-1. Our data revealed that by systematically mutating the key amino acid residues, we can modulate the amyloidogenic propensity and alter the time-dependent conformational variation of the peptide. When the behaviors of each mutant peptide were analyzed, they provided evidence consistent with the aggregation pathway predicted by MD simulation studies. Here, we detail the important temporal molecular interactions formed by Arg-1 with Ser-5, Glu-9, and Asp-12 and discuss its mechanistic implications on the self-assembly of the helix-1 region of SAA.

Identification of a membrane-associated element (MAE) in the C-terminal region of SARS-CoV-2 nsp6 that is essential for viral replication.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the novel coronavirus severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has rapidly spread worldwide since its emergence in late 2019. Its ongoing evolution poses challenges for antiviral drug development. Coronavirus nsp6, a multiple-spanning transmembrane protein, participates in the biogenesis of the viral replication complex, which accommodates the viral replication-transcription complex. The roles of its structural domains in viral replication are not well studied. Herein, we predicted the structure of the SARS-CoV-2 nsp6 protein using AlphaFold2 and identified a highly folded C-terminal region (nsp6C) downstream of the transmembrane helices. The enhanced green fluorescent protein (EGFP)-fused nsp6C was found to cluster in the cytoplasm and associate with membranes. Functional mapping identified a minimal membrane-associated element (MAE) as the region from amino acids 237 to 276 (LGV-KLL), which is mainly composed of the α-helix H1 and the α-helix H2; the latter exhibits characteristics of an amphipathic helix (AH). Mutagenesis studies and membrane flotation experiments demonstrate that AH-like H2 is required for MAE-mediated membrane association. This MAE was functionally conserved across MERS-CoV, HCoV-OC43, HCoV-229E, HCoV-HKU1, and HCoV-NL63, all capable of mediating membrane association. In a SARS-CoV-2 replicon system, mutagenesis studies of H2 and replacements of H1 and H2 with their homologous counterparts demonstrated requirements of residues on both sides of the H2 and properly paired H1-H2 for MAE-mediated membrane association and viral replication. Notably, mutations I266A and K274A significantly attenuated viral replication without dramatically affecting membrane association, suggesting a dual role of the MAE in viral replication: mediating membrane association as well as participating in protein-protein interactions.IMPORTANCESevere acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) assembles a double-membrane vesicle (DMV) by the viral non-structural proteins for viral replication. Understanding the mechanisms of the DMV assembly is of paramount importance for antiviral development. Nsp6, a multiple-spanning transmembrane protein, plays an important role in the DMV biogenesis. Herein, we predicted the nsp6 structure of SARS-CoV-2 and other human coronaviruses using AlphaFold2 and identified a putative membrane-associated element (MAE) in the highly conserved C-terminal regions of nsp6. Experimentally, we verified a functionally conserved minimal MAE composed of two α-helices, the H1, and the amphipathic helix-like H2. Mutagenesis studies confirmed the requirement of H2 for MAE-mediated membrane association and viral replication and demonstrated a dual role of the MAE in viral replication, by mediating membrane association and participating in residue-specific interactions. This functionally conserved MAE may serve as a novel anti-viral target.

Computationally efficient graph neural/convolutional network captures protein geometry to predict affinity and developability.

There are thousands, if not hundreds of thousands, of known proteins but a significantly lower number of unique protein folds. As a result, proteins often have conserved geometric patterns, including the ways amino acids cluster together. Delaunay tessellations have been successfully used in multiple methods involving protein structures, including applications to elastic network models to predict hinges and to predict conformational heterogeneity. In this work, we use Delaunay tessellations and alpha shapes to capture these conserved geometric patterns to define edges that are better suited to model protein packing than other established ways to form the edges for Graph Neural Network (GNN) architectures, including Graph Convolutional Networks (GCN). We test the resulting GCN models on problems including antibody affinity and developability. Compared to GNN architectures based on k-nearest neighbors or a fixed cutoff radius, models employing Delaunay tessellation can capture residue-specific interactions at a lower computational cost.

Bioinformatic prediction and experimental validation of RiPP recognition elements.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a family of natural products for which discovery efforts have rapidly grown over the past decade. There are currently 38 known RiPP classes encoded by prokaryotes. Half of the prokaryotic RiPP classes include a protein domain called the RiPP Recognition Element (RRE) for successful installation of post-translational modifications on a RiPP precursor peptide. In most cases, the RRE domain binds to the N-terminal "leader" region of the precursor peptide, facilitating enzymatic modification of the C-terminal "core" region. The prevalence of the RRE domain renders it a theoretically useful bioinformatic handle for class-independent RiPP discovery; however, first-in-class RiPPs have yet to be isolated and experimentally characterized using an RRE-centric strategy. Moreover, with most known RRE domains engaging their cognate precursor peptide(s) with high specificity and nanomolar affinity, evaluation of the residue-specific interactions that govern RRE:substrate complexation is a necessary first step to leveraging the RRE domain for various bioengineering applications. This chapter details protocols for developing custom bioinformatic models to predict and annotate RRE domains in a class-specific manner. Next, we outline methods for experimental validation of precursor peptide binding using fluorescence polarization binding assays and in vitro enzyme activity assays. We anticipate the methods herein will guide and enhance future critical analyses of the RRE domain, eventually enabling its future use as a customizable tool for molecular biology.

NMR Methods for Characterization of Glycosaminoglycan-Chemokine Interactions.

Humans express around 50 chemokines that play crucial roles in human pathophysiology from combating infection to immune surveillance by directing and trafficking leukocytes to the target tissue. Glycosaminoglycans (GAGs) regulate chemokine function by tuning monomer/dimer levels, chemotactic/haptotactic gradients, and how they are presented to their receptors. Knowledge of the structural features of the chemokine-GAG complexes and GAG properties that define chemokine interactions is essential not only to understand chemokine function, but also for developing drugs that disrupt chemokine-GAG crosstalk and thereby impart protection against dysregulated host defense. Nuclear magnetic resonance (NMR) spectroscopy has proven to be quite useful for providing residue-specific interactions, binding geometry and models, specificity, and affinity. Multiple NMR methods have been used including (1) chemical shift perturbation (CSP), (2) saturation transfer difference (STD), and (3) paramagnetic relaxation enhancement (PRE) techniques. In this chapter, we describe how NMR CSP, STD, and PRE can be best used for characterizing chemokine-GAG interactions.

Analysis of Global and Residue‐Specific yB Crystallin Protein Dynamics via NMR Spectroscopy and Dynamic Light Scattering

The proteins that comprise the water‐soluble interior of the optic lens are referred to as crystallin proteins, of which three major categories have been identified: alpha (a), beta (b), and gamma (y). Gamma and beta crystallin proteins have been observed to undergo aggregation and phase‐separation over time, which can lead to the formation of cataracts, while alpha crystallin limits this formation by removing deformed protein aggregates from solution. Here, we describe our studies on bovine yB crystallin, which is the homologue to human yD crystallin. To better understand the intermolecular interactions between yB crystallins, we overexpressed the 15N‐labeled recombinant protein in Escherichia coli, purified it via column chromatography, and studied the effect of solution conditions on protein dynamics using NMR spectroscopy and dynamic light scattering techniques. Prior research from our group employed T1‐T2 NMR experiments to probe the effect of temperature and protein concentration on global protein dynamics, while more recent 1H‐15N HSQC experiments allowed us to visualize the amide proton peaks for yB crystallin protein at various concentrations. We observed small peak shifts for select residues as the protein concentration increased, which we hypothesized were due to increased intermolecular interactions between yB crystallin molecules. Analysis of these NMR spectra, combined with dynamic light scattering data on the same samples, provide further insight into both global dynamics as well as residue‐specific interactions.

Mutational Effect of Some Major COVID-19 Variants on Binding of the S Protein to ACE2.

COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has many variants that accelerated the spread of the virus. In this study, we investigated the quantitative effect of some major mutants of the spike protein of SARS-CoV-2 binding to the human angiotensin-converting enzyme 2 (ACE2). These mutations are directly related to the Variant of Concern (VOC) including Alpha, Beta, Gamma, Delta and Omicron. Our calculations show that five major mutations (N501Y, E484K, L452R, T478K and K417N), first reported in Alpha, Beta, Gamma and Delta variants, all increase the binding of the S protein to ACE2 (except K417N), consistent with the experimental findings. We also studied an additional eight mutations of the Omicron variant that are located on the interface of the receptor binding domain (RDB) and have not been reported in other VOCs. Our study showed that most of these mutations (except Y505H and G446S) enhance the binding of the S protein to ACE2. The computational predictions helped explain why the Omicron variant quickly became dominant worldwide. Finally, comparison of several different computational methods for binding free energy calculation of these mutants was made. The alanine scanning method used in the current calculation helped to elucidate the residue-specific interactions responsible for the enhanced binding affinities of the mutants. The results show that the ASGB (alanine scanning with generalized Born) method is an efficient and reliable method for these binding free energy calculations due to mutations.

Investigating novel thiazolyl-indazole derivatives as scaffolds for SARS-CoV-2 MPro inhibitors

COVID-19 is a global pandemic caused by infection with the SARS-CoV-2 virus. Remdesivir, a SARS-CoV-2 RNA polymerase inhibitor, is the only drug to have received widespread approval for treatment of COVID-19. The SARS-CoV-2 main protease enzyme (MPro), essential for viral replication and transcription, remains an active target in the search for new treatments. In this study, the ability of novel thiazolyl-indazole derivatives to inhibit MPro is evaluated. These compounds were synthesized via the heterocyclization of phenacyl bromide with (R)-carvone, (R)-pulegone and (R)-menthone thiosemicarbazones. The binding affinity and binding interactions of each compound were evaluated through Schrödinger Glide docking, AMBER molecular dynamics simulations, and MM-GBSA free energy estimation, and these results were compared with similar calculations of MPro binding various 5-mer substrates (VKLQA, VKLQS, VKLQG) and a previously identified MPro tight-binder X77. From these simulations, we can see that binding is driven by residue specific interactions such as π-stacking with His41, and S/π interactions with Met49 and Met165. The compounds were also experimentally evaluated in a MPro biochemical assay and the most potent compound containing a phenylthiazole moiety inhibited protease activity with an IC50 of 92.9 ​μM. This suggests that the phenylthiazole scaffold is a promising candidate for the development of future MPro inhibitors.

Residue-by-residue analysis of cotranslational membrane protein integration in vivo.

We follow the cotranslational biosynthesis of three multispanning Escherichia coli inner membrane proteins in vivo using high-resolution force profile analysis. The force profiles show that the nascent chain is subjected to rapidly varying pulling forces during translation and reveal unexpected complexities in the membrane integration process. We find that an N-terminal cytoplasmic domain can fold in the ribosome exit tunnel before membrane integration starts, that charged residues and membrane-interacting segments such as re-entrant loops and surface helices flanking a transmembrane helix (TMH) can advance or delay membrane integration, and that point mutations in an upstream TMH can affect the pulling forces generated by downstream TMHs in a highly position-dependent manner, suggestive of residue-specific interactions between TMHs during the integration process. Our results support the 'sliding' model of translocon-mediated membrane protein integration, in which hydrophobic segments are continually exposed to the lipid bilayer during their passage through the SecYEG translocon.

Molecular Details of a Salt Bridge and Its Role in Insulin Fibrillation by NMR and Raman Spectroscopic Analysis.

Insulin, a simple polypeptide hormone with huge biological importance, has long been known to self-assemble in vitro and form amyloid-like fibrillar aggregates. Utilizing high-resolution NMR, Raman spectroscopy, and computational analysis, we demonstrate that the fluctuation of the carboxyl terminal (C-ter) residues of the insulin B-chain plays a key role in the growth phase of insulin aggregation. By comparing the insulin sourced from bovine, human, and the modified glargine (GI), we observed reduced aggregation propensity in the GI variant, resulting from two additional Arg residues at its C-ter. NMR analysis showed atomic contacts and residue-specific interactions, particularly the salt bridge and H-bond formed among the C-ter residues Arg31B, Lys29B, and Glu4A. These inter-residue interactions were reflected in strong nuclear Overhauser effects among Arg31BδH-Glu4AδH and Lys29BδHs-Glu4AδH in GI, as well as the associated downfield chemical shift of several A-chain amino terminal (N-ter) residues. The two additional Arg residues of GI, Arg31B and Arg32B, enhanced the stability of the GI native structure by strengthening the Arg31B, Lys29B, and Glu4A salt bridge, thus reducing extensive thermal distortion and fluctuation of the terminal residues. The high stability of the salt bridge retards tertiary collapse, a crucial biochemical event for oligomerization and subsequent fibril formation. Circular dichroism and Raman spectroscopic measurement also suggest slow structural distortion in the early phase of the aggregation of GI because of the restricted mobility of the C-ter residues as explained by NMR. In addition, the structural and dynamic parameters derived from molecular dynamics simulations of insulin variants highlight the role of residue-specific contacts in aggregation and amyloid-like fibril formation.

Energy Decomposition Analysis of Protein-Ligand Interactions Using Molecules-in-Molecules Fragmentation-Based Method.

Accurate prediction of protein-ligand binding affinities and their quantitative decomposition into residue-specific contributions represent challenging problems in drug discovery. While quantum mechanical (QM) methods can provide an accurate description of such interactions, the associated computational cost is normally prohibitive for broad-based applications. Recently, we have shown that QM-based protein-ligand interaction energies in the gas phase can be determined accurately using our multilayer molecules-in-molecules (MIM) fragmentation-based method at a significantly lower computational cost. In this paper, we present a new approach for calculating protein-ligand interactions using our three-layer model (MIM3) that allows us to decompose the total binding affinity into quantitative contributions from individual residues (or backbone and side chain), crystal water molecules, solvation energy, and entropy. In our approach, the desolvation energy and entropy changes during protein-ligand binding are modeled using simple and inexpensive empirical models while intermolecular interactions are computed using an accurate QM method. The performance of our approach has been assessed on a congeneric series of 22 thrombin inhibitors, all with experimentally known binding affinities, using a binding pocket cutout of 120 residues with more than 1550 atoms. Comparison of our MIM3-calculated binding affinities calculated at the B97-D3BJ/6-311++G(2d,2p) level with experiment shows a good correlation with an R2 range of 0.81-0.88 and a Spearman rank correlation coefficient (ρ) range of 0.84-0.89 while providing a quantitative description of residue-specific interactions. We show that such residue-specific interaction energies can be employed to identify and rationalize both obvious (e.g., hydrogen bonds, π···π) and nonobvious (e.g., CH···π) interactions that play a critical role in protein-ligand binding. We suggest that such quantitative information can be used to identify the key residues that determine the comparative binding affinities of different ligands in order to improve and optimize the effectiveness of computational drug design.

Understanding the Structural Basis of Recognition between Plasmodium Falciparum and Human Sumoylation Machinery

Malaria is one of the deadliest infectious diseases, affecting millions of lives annually and is caused by parasitic protozoans of the genus Plasmodium. P. falciparum (Pf) adopts various survival strategies including post-translational modifications (PTMs) to stabilize and potentiate its crucial proteins for successful infection cycle in the mosquito and human host. One of the important PTMs is called sumoylation, which is essential for normal cellular functions. It is known that SUMO interacts with both E1-activating (hetero-dimeric Aos1/Uba2), and E2-conjugating (Ubc9) individually. Can the interaction of Pf-SUMO with E1and/or E2 have the cross-species interaction element that can be targeted? Moreover, the structure of Pf-SUMO is not known, we present the first structure of Pf-SUMO solved using solution state NMR. The residue specific interactions of Pf-SUMO with Pf-Ubc9 and the possibilities of cross-interaction of host and parasite sumoylation machineries were also checked by NMR titration studies. We have identified the important residues of Pf-SUMO proteins involved in sumoylation that governs the specificity of interaction in Plasmodium as well as human host. The residues at the interacting interface that displayed prominent interactions have been mutated to probe their specificity. Overall, all these results emphasize that during disease condition the parasite may use the host sumoylation machinery to maintain its survival. This information can be used for designing drugs that specifically block the interface. The dynamics of Pf-SUMO protein will also help in ascertaining role of protein plasticity in substrate recognition and specificity.

Residue-Specific Interactions of an Intrinsically Disordered Protein with Silica Nanoparticles and their Quantitative Prediction.

Elucidation of the driving forces that govern interactions between nanoparticles and intrinsically disordered proteins (IDP) is important for the understanding of the effect of nanoparticles in living systems and for the design of new nanoparticle-based assays to monitor health and combat disease. The quantitative interaction profile of the intrinsically disordered transactivation domain of p53 and its mutants with anionic silica nanoparticles is reported at atomic resolution using nuclear magnetic spin relaxation experiments. These profiles are analyzed with a novel interaction model that is based on a quantitative nanoparticle affinity scale separately derived for the 20 natural amino acids. The results demonstrate how the interplay of attractive and repulsive Coulomb interactions with hydrophobic effects is responsible for the sequence-dependent binding of a disordered protein to nanoparticles.

Protein-dependent Membrane Interaction of A Partially Disordered Protein Complex with Oleic Acid: Implications for Cancer Lipidomics.

Bovine α-lactalbumin (BLA) forms cytotoxic complexes with oleic acid (OA) that perturbs tumor cell membranes, but molecular determinants of these membrane-interactions remain poorly understood. Here, we aim to obtain molecular insights into the interaction of BLA/BLA-OA complex with model membranes. We characterized the folding state of BLA-OA complex using tryptophan fluorescence and resolved residue-specific interactions of BLA with OA using molecular dynamics simulation. We integrated membrane-binding data using a voltage-sensitive probe and molecular dynamics (MD) to demonstrate the preferential interaction of the BLA-OA complex with negatively charged membranes. We identified amino acid residues of BLA and BLA-OA complex as determinants of these membrane interactions using MD, functionally corroborated by uptake of the corresponding α-LA peptides across tumor cell membranes. The results suggest that the α-LA component of these cytotoxic complexes confers specificity for tumor cell membranes through protein interactions that are maintained even in the lipid complex, in the presence of OA.

Rotation of artificial rotor axles in rotary molecular motors

F1- and V1-ATPase are rotary molecular motors that convert chemical energy released upon ATP hydrolysis into torque to rotate a central rotor axle against the surrounding catalytic stator cylinder with high efficiency. How conformational change occurring in the stator is coupled to the rotary motion of the axle is the key unknown in the mechanism of rotary motors. Here, we generated chimeric motor proteins by inserting an exogenous rod protein, FliJ, into the stator ring of F1 or of V1 and tested the rotation properties of these chimeric motors. Both motors showed unidirectional and continuous rotation, despite no obvious homology in amino acid sequence between FliJ and the intrinsic rotor subunit of F1 or V1 These results showed that any residue-specific interactions between the stator and rotor are not a prerequisite for unidirectional rotation of both F1 and V1 The torque of chimeric motors estimated from viscous friction of the rotation probe against medium revealed that whereas the F1-FliJ chimera generates only 10% of WT F1, the V1-FliJ chimera generates torque comparable to that of V1 with the native axle protein that is structurally more similar to FliJ than the native rotor of F1 This suggests that the gross structural mismatch hinders smooth rotation of FliJ accompanied with the stator ring of F1.

On the domains of T4 phage sliding clamp gp45: An intermolecular crosstalk governs structural stability and biological activity

BackgroundDNA polymerase processivity factors are ubiquitously present in all living organisms. Notwithstanding their high significance, the molecular details of clamps pertaining to the factors contributing to their stability are presently lacking. The bacteriophage T4 sliding clamp gp45 forms a homotrimer that besides being involved in DNA replication, moonlights as a transcription factor. Here we have carried out a detailed characterization of gp45 to understand the role of monomer-monomer interface interactions in stability and functioning of the protein. MethodsWe generated several gp45 mutants harboring either Ala or Pro substitutions at the interface residues and performed a detailed investigation using biochemical and biophysical methods including circular dichroism, fluorescence anisotropy and quenching, differential scanning calorimetry, blue-native PAGE, cross-linking, size exclusion chromatography, and dynamic light scattering. We also carried out both transcription and DNA replication to understand the properties of the wild-type and the mutant proteins. ResultsOne specific mutation S88P leads not only to monomerization, but also results in an unstable molecule. Most interestingly, mutating either Q125 or K164 in the gp45 C-terminal domain negatively affects the stability of the N-terminal domain. We also report that these residues upon mutation to alanine make gp45 inactive for late promoter transcription, whereas strand-displacement DNA replication ability remains unaltered. Conclusions and general significanceThe results suggest that the two domains of gp45 demonstrate an “inter-monomer” crosstalk that stabilizes the trimer. We also conclude that the residue-specific interactions at the interface allow the protein to function distinctly as replication and transcription factors.

NMR Characterization and Membrane Interactions of the Loop Region of Kindlin-3 F1 Subdomain.

Kindlins-1,2 and 3 are FERM domain-containing cytosolic proteins involved in the activation and regulation of integrin-mediated cell adhesion. Apart from binding to integrin β cytosolic tails, kindlins and the well characterized integrin-activator talin bind membrane phospholipids. The ubiquitin-like F1 sub-domain of the FERM domain of talin contains a short loop that binds to the lipid membrane. By contrast, the F1 sub-domain of kindlins contains a long loop demonstrated binding to the membrane. Here, we report structural characterization and lipid interactions of the 83-residue F1 loop of kindlin-3 using NMR and optical spectroscopy methods. NMR studies demonstrated that the F1 loop of kindlin-3 is globally unfolded but stretches of residues assuming transient helical conformations could be detected in aqueous solution. We mapped membrane binding interactions of the F1 loop with small unilamellar vesicles (SUVs) containing either zwitterionic lipids or negatively charged lipids using 15N-1H HSQC titrations. These experiments revealed that the F1 loop of kindlin-3 preferentially interacted with the negatively charged SUVs employing almost all of the residues. By contrast, only fewer residues appeared to be interacted with SUVs containing neutral lipids. Further, CD and NMR data suggested stabilization of helical conformations and predominant resonance perturbations of the F1 loop in detergent containing solutions. Conformations of an isolated N-terminal peptide fragment, or EK21, of the F1 loop, containing a poly-Lys sequence motif, important for membrane interactions, were also investigated in detergent solutions. EK21 adopted a rather extended or β-type conformations in complex with negatively charged SDS micelles. To our knowledge, this is the first report describing the conformations and residue-specific interactions of kindlin F1 loop with lipids. These data therefore provide important insights into the interactions of kindlin FERM domain with membrane lipids that contribute toward the integrin activating property.

Muscle Shortening Velocity is Modulated by Alternative Myosin Converters

The myosin converter has been proposed to be critical for setting muscle shortening velocity by influencing the amplification of small conformational changes at the ATPase site into the larger lever arm swing. To test this hypothesis, we exploited the natural variation of the converter region found in Drosophila muscle types. In Drosophila, a single myosin heavy chain gene is alternatively spliced to generate myosin isoforms. The converter region is encoded by five alternative versions of exon 11 that are expressed in different muscle types. Through genetic modification we forced the expression of the alternative versions in the Drosophila jump muscle and found that two of the three versions tested to date, 11a (native to indirect flight muscles) and 11e (embryonic muscles), caused faster maximum shortening velocities of skinned jump muscle fibers relative to the native control 11c. Increases in velocity were primarily responsible for 70.9% and 79.0% higher power outputs for 11a and 11e fibers, respectively, because maximum force generation was not significantly different from control fibers. The fibers expressing 11a and 11e also exhibited a straighter force-velocity curve, as shown by increased Hill equation parameters a (95.4% and 84.5% higher, respectively) and b (72.6% and 65.5% higher, respectively) relative to control fibers, indicating that the converter modulates load dependent cross-bridge kinetics. The higher power and shortening velocity of 11a and 11e fibers enabled increased jumping ability, 76.0% and 48.8% farther, respectively. Converter homology models, using the new Drosophila myosin S1 crystal structure (pdb 4QBD), revealed minimal tertiary structure differences suggesting residue specific interactions with the relay, N-terminus, or essential light chain are critical. Overall, our data support the hypothesis that the converter helps set muscle shortening velocity under loaded and unloaded conditions and thus contributes to muscle fiber type diversity.

Energy landscapes reveal the myopathic effects of tropomyosin mutations

Striated muscle contraction is regulated by an interaction network connecting the effects of troponin, Ca2+, and myosin-heads to the azimuthal positioning of tropomyosin along thin filaments. Many missense mutations, located at the actin–tropomyosin interface, however, reset the regulatory switching mechanism either by weakening or strengthening residue-specific interactions, leading to hyper- or hypo-contractile pathologies. Here, we compute energy landscapes for the actin–tropomyosin interface and quantify contributions of single amino acid residues to actin–tropomyosin binding. The method is a useful tool to assess effects of actin and tropomyosin mutations, potentially relating initial stages of myopathy to alterations in thin filament stability and regulation. Landscapes for mutant filaments linked to hyper-contractility provide a simple picture that describes a decrease in actin–tropomyosin interaction energy. Destabilizing the blocked (relaxed)-state parallels previously noted enhanced Ca2+-sensitivity conferred by these mutants. Energy landscapes also identify post-translational modifications that can rescue regulatory imbalances. For example, cardiomyopathy-associated E62Q tropomyosin mutation weakens actin–tropomyosin interaction, but phosphorylation of neighboring S61 rescues the binding-deficit, results confirmed experimentally by in vitro motility assays. Unlike results on hyper-contractility-related mutants, landscapes for tropomyosin mutants tied to hypo-contractility do not present a straightforward picture. These mutations may affect other components of the regulatory network, e.g., troponin–tropomyosin signaling.

Role of N-terminal residues in Aβ interactions with integrin receptor and cell surface

beta-Amyloid (Aβ) is the primary protein component of senile plaques in Alzheimer's disease (AD) and is believed to play a role in its pathology. To date, the mechanism of action of Aβ in AD is unclear. We and others have observed that Aβ interacts either with or in the vicinity of the α6 sub-unit of integrin, and believe this may be important in its interaction with neuronal cells. In this study, we used confocal microscopy and flow cytometry to explore the residue specific interactions of Aβ40 with the cell surface and the α6 integrin receptor sub-unit. We probed the importance of the RHD sequence in Aβ40 and found that removal of the residues or their mutation using the Aβ8-40 or the D7N early onset AD sequence, respectively, led to a greater interaction between Aβ40 and an antibody bound to the α6-integrin sub-unit, as measured by fluorescence resonance energy transfer (FRET). These results suggest that the RHD sequence of Aβ40 does not mediate Aβ–α6 integrin interactions. However, the cyclic RGD mimicking peptide, Cilengitide, reduced the measured interaction between Aβ40 fibrils without the RHD sequence and an antibody bound to the α6-integrin sub-unit. We further probed the role of electrostatic forces on Aβ40–cell interactions and observed that the Aβ sequence that included the N-terminal segment of the peptide had reduced cellular binding at low salt concentrations, suggesting that its first 7 residues contribute to an electrostatic repulsion for the cell surface. These findings contribute to our understanding of Aβ–cell surface interactions and may provide insight into development of novel strategies to block Aβ–cell interactions that contribute to pathology in Alzheimer's disease.