αB-crystallin is a small heat shock protein that forms a heterooligomeric complex with αA-crystallin in the ocular lens. It is also widely distributed in tissues throughout the body and has been linked with neurodegenerative diseases such as Alzheimer’s, where it is associated with amyloid fibrils. Crystallins can form amorphous aggregates in cataracts as well as more structured amyloid-like fibrils. The arginine 120 to glycine (R120G) mutation in αB-crystallin ( Cryab -R120G) results in high molecular weight crystallin protein aggregates and loss of the chaperone activity of the protein in vitro , and it is associated with human hereditary cataracts and myopathy. Characterizing the amorphous (unstructured) versus the highly ordered (amyloid fibril) nature of crystallin aggregates is important in understanding their role in disease and important to developing pharmacological treatments for cataracts. We investigated protein secondary structure in wild-type (WT) and Cryab -R120G knock-in mutant mouse lenses using two-dimensional infrared (2DIR) spectroscopy, which has been used to detect amyloid-like fibrils in human lenses and measure UV radiation-induced changes in porcine lenses. Our goal was to compare the aggregated proteins in this mouse lens model to human lenses and evaluate the protein structural relevance of the Cryab -R120G knock-in mouse model to general age-related cataract disease. In the 2DIR spectra, amide I diagonal peak frequencies were red-shifted to smaller wavenumbers in mutant mouse lenses as compared to WT mouse lenses, consistent with an increase in ordered secondary structure. The cross peak frequency and intensity indicated the presence of amyloid in the mutant mouse lenses. While the diagonal and cross peak changes in location and intensity from the 2DIR spectra indicated significant structural differences between the wild type and mutant mouse lenses, these differences were smaller than those found in human lenses; thus, the Cryab -R120G knock-in mouse lenses contain less amyloid-like secondary structure than human lenses. The results of the 2DIR spectroscopy study confirm the presence of amyloid-like secondary structure in Cryab -R120G knock-in mice with cataracts and support the use of this model to study age-related cataract.

Transmembrane signaling proteins couple extracytosolic sensors to cytosolic effectors. Here, we examine how binding of Mg 2+ to the sensor domain of an E. coli two component histidine kinase (HK), PhoQ, modulates its cytoplasmic kinase domain. We use cysteine-crosslinking and reporter-gene assays to simultaneously and independently probe the signaling state of PhoQ’s sensor and autokinase domains in a set of over 30 mutants. Strikingly, conservative single-site mutations distant from the sensor or catalytic site strongly influence PhoQ’s ligand-sensitivity as well as the magnitude and direction of the signal. Data from 35 mutants are explained by a semi-empirical three-domain model in which the sensor, intervening HAMP, and catalytic domains can adopt kinase-promoting or inhibiting conformations that are in allosteric communication. The catalytic and sensor domains intrinsically favor a constitutively ‘kinase-on’ conformation, while the HAMP domain favors the ‘off’ state; when coupled, they create a bistable system responsive to physiological concentrations of Mg 2+ . Mutations alter signaling by locally modulating domain intrinsic equilibrium constants and interdomain couplings. Our model suggests signals transmit via interdomain allostery rather than propagation of a single concerted conformational change, explaining the diversity of signaling structural transitions observed in individual HK domains.

Blood clot contraction is driven by traction forces generated by the platelet cytoskeleton that are transmitted to fibrin fibers via the integrin αIIbβ3. Here we show that clot contraction is impaired by inhibitors of the platelet cytosolic protease calpain. We used subtiligase-mediated labeling of amino termini and mass spectrometry to identify proteolytically cleaved platelet proteins involved in clot contraction. Of 32 calpain-cleaved proteins after TRAP stimulation, 14 were cytoskeletal, most prominently talin and vinculin. A complex of talin and vinculin constitutes a mechanosensitive clutch connecting integrins bound to the extracellular matrix with the actin cytoskeleton. Accordingly, we focused on talin and vinculin. Talin is composed of an N-terminal head domain and a C-terminal rod domain organized into a series of 4- and 5-helix bundles. The bundles contain 11 vinculin binding sites (VBSs), each of which is an α-helix packed into a bundle interior and requiring structural rearrangement to initiate vinculin binding. We detected 8 calpain-mediated cleavages in talin, 2 previously identified in unstructured regions and 6 in α-helical regions in proximity to a VBS. There is evidence in vitro that applying mechanical force across talin enables vinculin binding to the talin rod. However, we found that inhibiting platelet cytoskeletal contraction had no effect on talin cleavage, indicating that talin cleavage by calpain in platelets does not require cytoskeleton-generated tensile force. Therefore, it is likely that calpain acts in the later stages of clot retraction through focal adhesion disassembly.

Fibrillins serve as scaffolds for the assembly of elastic fibers that contribute to the maintenance of tissue homeostasis and regulate growth factor signaling in the extracellular space. Fibrillin-1 is a modular glycoprotein that includes 7 latent transforming growth factor ? (TGF?)-binding protein-like (TB) domains and mediates cell adhesion through integrin binding to the RGD motif in its 4th TB domain. A subset of missense mutations within TB4 cause stiff skin syndrome (SSS), a rare autosomal dominant form of scleroderma. The fibrotic phenotype is thought to be regulated by changes in the ability of fibrillin-1 to mediate integrin binding. We characterized the ability of each RGD-binding integrin to mediate cell adhesion to fibrillin-1 or a disease-causing variant. Our data show that 7 of the 8 RGD-binding integrins can mediate adhesion to fibrillin-1. A single amino acid substitution responsible for SSS (W1570C) markedly inhibited adhesion mediated by integrins ?5?1, ?v?5, and ?v?6, partially inhibited adhesion mediated by ?v?1, and did not inhibit adhesion mediated by ?8?1 or ?IIb?3. Adhesion mediated by integrin ?v?3 depended on the cell surface expression level. In the SSS mutant background, the presence of a cysteine residue in place of highly conserved tryptophan 1570 alters the conformation of the region containing the exposed RGD sequence within the same domain to differentially affect fibrillin's interactions with distinct RGD-binding integrins.

Purpose:We previously identified an oxysterol, VP1-001 (also known as compound 29), that partially restores the transparency of lenses with cataracts. To understand the mechanism of VP1-001, we tested the ability of its enantiomer, ent-VP1-001, to bind and stabilize αB-crystallin (cryAB) in vitro and to produce a similar therapeutic effect in cryAB(R120G) mutant and aged wild-type mice with cataracts. VP1-001 and ent-VP1-001 have identical physicochemical properties. These experiments are designed to critically evaluate whether stereoselective binding to cryAB is required for activity.Methods:We compared the binding of VP1-001 and ent-VP1-001 to cryAB using in silico docking, differential scanning fluorimetry (DSF), and microscale thermophoresis (MST). Compounds were delivered by six topical administrations to mouse eyes over 2 weeks, and the effects on cataracts and lens refractive measures in vivo were examined. Additionally, lens epithelial and fiber cell morphologies were assessed via transmission electron microscopy.Results:Docking studies suggested greater binding of VP1-001 into a deep groove in the cryAB dimer compared with ent-VP1-001. Consistent with this prediction, DSF and MST experiments showed that VP1-001 bound cryAB, whereas ent-VP1-001 did not. Accordingly, topical treatment of lenses with ent-VP1-001 had no effect, whereas VP1-001 produced a statistically significant improvement in lens clarity and favorable changes in lens morphology.Conclusions:The ability of VP1-001 to bind native cryAB dimers is important for its ability to reverse lens opacity in mouse models of cataracts.

Program Number: 5298 Poster Board Number: B0266 Presentation Time: 8:30 AM–10:15 AM Use of CRISPR/Cas9 to assess the role of αA-crystallin in zebrafish lens development Mason Posner, Kelly L. Murray, Mary Brown. Biology and Toxicology, Ashland University, Ashland, OH. Purpose: We previously reported that morpholino (MO) knockdown of αA-crystallin does not cause abnormalities in the zebrafish lens. However, another study found noticeable lens defects after MO knockdown and a more subtle lens phenotype in a TALEN induced mutant line. We have now used CRISPR/Cas9 to generate two αAcrystallin zebrafish mutant lines to assess the effect of the protein’s loss. Methods: We generated guide RNA (gRNA) to direct Cas9 cleavage within exon 1 of zebrafish αA-crystallin. Purified gRNA (455 pg) was coinjected with Cas9 protein (150 pg) into zebrafish zygotes using a total volume of 1 nl. Possible mutations at the target site were assessed using a T7 endonuclease assay on genomic DNA from pooled embryos. Individual fish from successful injections were genotyped by sequencing the target area from tail clip genomic DNA. Founder fish were crossed to ZDR strain wild-type adults to generate an F1 heterozygous population, which was then incrossed to check for potential phenotypes. Resulting embryos were PTU treated and examined by DIC for lens defects or fixed for histological examination. F1 fish were also individually genotyped to confirm the expected mutation and used to produce stable lines for future studies. Results: We generated two fish with 7 and 5 basepair deletions that led to an early stop codon after 29 and 30 amino acids, respectively. Embryos from an F1 incross of the 7-basepair deletion founder were generated to identify possible phenotypes. Out of 36 embryos examined at 5 days post fertilization (dpf) two had minor lens defects manifested as central irregularities viewed by DIC imaging. Of twelve embryos examined by sectioning and H&E staining six showed some lens shape irregularity, although fiber cell differentiation appeared normal. Conclusions: We observed some possible effects of αA-crystallin loss on lens development at 5 dpf. The lack of any noticeable phenotype in our previous αA MO knockdowns through 4 dpf may suggest that only small amounts of this protein are required for normal lens development, or that lens phenotypes do not appear until after day 4. The prevalence, severity and time course of any phenotype will be quantified by examination of additional embryos from our two generated knockout lines. These data highlight the usefulness of using the CRISPR/Cas9 system to efficiently delete lens crystallins for the study of development and age-related cataracts. Commercial Relationships: Mason Posner; Kelly L. Murray, None; Mary Brown, None Support: NEI Grant R15EY013535

One small molecule inhibitor of αvβ1 integrin, c8, shows antifibrotic effects in multiple in vivo mouse models. Here we synthesized c8 analogues and systematically investigate their structure-activity relationships (SAR) in αvβ1 integrin inhibition. N-Phenylsulfonyl-l-homoproline analogues of c8 maintained excellent potency against αvβ1 integrin while retaining good selectivity over other RGD integrins. In addition, 2-aminopyridine or cyclic guanidine analogues were shown to be equally potent to c8. A rigid phenyl linker increased the potency compared to c8, but the selectivity over other RGD integrins diminished. These results can provide further insights on design of αvβ1 integrin inhibitors as antifibrotics.

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.

This directory contains the input data, protocols and output model for the modeling of the PhoQ homodimer, using cysteine crosslinking and multi-state Bayesian modeling in IMP. For more information about how to reproduce this modeling, see https://salilab.org/phoq or the README file.

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.

Abstract Abstract 1255 Kindlins are a family of FERM domain proteins that are essential for inside-out integrin activation. In particular, kindlin-3 is required for the conversion of the major platelet integrin αIIbβ3 from its resting conformation to its active ligand binding conformation. Moreover, naturally-occurring kindlin-3 mutations result in the inherited disorder leukocyte adhesion deficiency III, one component of which is defective platelet function that mimics Glanzmann thrombasthenia. Despite the importance of kindlin-3 in initiating αIIbβ3 function in platelets, little is known about its regulation in resting platelets or its fate in activated platelets. To address these questions, we purified full-length kindlin-3 from outdated human platelets where it is present in substantial amounts and also developed a procedure to synthesize substantial amounts of recombinant kindlin-3 in SF9 cells. We found that in stored human platelets, kindlin-3 is cleaved into two fragments as a function of the time of storage. We also found that kindlin-3 is cleaved into identical fragments when fresh human platelets are stimulated with the thrombin receptor activating peptide (TRAP) for 5 minutes. To identify the site of kindlin-3 cleavage, as well as the responsible protease, we used a proteomics method in which an engineered peptide ligase, subtiligase, was used to selectively biotinylate the unblocked α amines of proteins obtained from platelet lysates (Mahrus et al, Cell 134:866–76, 2008). Biotinylated proteins were digested with trypsin and the resulting biotinylated peptide fragments were then captured using avidin agarose and identified using tandem mass spectrometry. Using this method, we identified the kindlin-3 peptide (G)SAPTDVLDSLTTIPELKDHL in lysates of stored platelets, thereby mapping the cleavage site to residues 335–336. We obtained identical results using proteins isolated from TRAP-stimulated platelets. Further, we were able to recapitulate these results in vitro using purified kindlin-3 and the calcium-activated protease calpain, implying that calpain is the responsible protease in vivo. Kindlin-3 is thought to initiate αIIbβ3 function by binding to the distal NITY motif in the β3 cytosolic tail in an interaction that also involves S752. Previously, we reported a model for αIIbβ3 regulation based on an NMR structure of the β3 cytosolic tail (Metcalf et al, PNAS 107:24775–83, 2010). In this structure, the NITY motif is located in a distal dynamic amphiphilic helix where the motif is transiently masked by interacting with the membrane. To validate this model, we have studied the interaction of both purified and recombinant kindlin-3 with the β3 tail using surface plasmon resonance (SPR). A peptide corresponding to β3 residues 719–762, encompassing the complete β3 tail, was immobilized on a CM5 chip and kindlin-3 was flowed over the chip surface. The resulting sensorgrams could then be fit to two binding events with dissociation constants of 2.2 nM and 2.8 μM. The biphasic behavior could have resulted from heterogeneity of the β3 tail on the chip surface or heterogeneity of the interaction between kindlin-3 and the carboxymethylated dextran. Similar SPR experiments measuring binding of the talin-1 FERM domain to the β3 tail also could be fit to two binding events with dissociation constants of 155 nM and 3.5 μM. Thus, under these experimental conditions, kindlin-3 binds approximately 70-fold more tightly to the β3 tail than does the talin-1 FERM domain. In summary, these studies demonstrate that kindlin-3 undergoes calpain-mediated endoproteolysis during platelet storage, an event that may contribute to the development of the platelet storage lesion. Kindlin-3 also undergoes an identical cleavage following platelet stimulation by agonists such as thrombin. Since high affinity binding of kindlin-3 to the β3 cytosolic tail is required for physiologic αIIbβ3 activation, it is possible that kindlin-3 cleavage attenuates αIIbβ3 activity. Disclosures: No relevant conflicts of interest to declare.

The integrin αIIbβ3 is a transmembrane (TM) heterodimeric adhesion receptor that exists in equilibrium between resting and active ligand binding conformations. In resting αIIbβ3, the TM and cytoplasmic domains of αIIb and β3 form a heterodimer that constrains αIIbβ3 in its resting conformation. To study the structure and dynamics of the cytoplasmic domain heterodimer, we prepared a disulfide-stabilized complex consisting of portions of the TM domains and the full cytoplasmic domains. NMR and hydrogen-deuterium exchange of this complex in micelles showed that the αIIb cytoplasmic domain is largely disordered, but it interacts with and influences the conformation of the β3 cytoplasmic domain. The β3 cytoplasmic domain consists of a stable proximal helix contiguous with the TM helix and two distal amphiphilic helices. To confirm the NMR structure in a membrane-like environment, we studied the β3 cytoplasmic domain tethered to phospholipid bilayers. Hydrogen-deuterium exchange mass spectrometry, as well as circular dichroism spectroscopy, demonstrated that the β3 cytoplasmic domain becomes more ordered and helical under these conditions, consistent with our NMR results. Further, these experiments suggest that the two distal helices associate with lipid bilayers but undergo fluctuations that would allow rapid binding of cytoplasmic proteins regulating integrin activation, such as talin and kindlin-3. Thus, these results provide a framework for understanding the kinetics and thermodynamics of protein interactions involving integrin cytoplasmic domains and suggest that such interactions act in a concerted fashion to influence integrin stalk separation and exposure of extracellular ligand binding sites.

ABSTRACT The binding reaction of the HIV-1 gp120 envelope glycoprotein to the CD4 receptor involves exceptional changes in enthalpy and entropy. Crystal structures of gp120 in unliganded and various ligand-bound states, meanwhile, reveal an inner domain able to fold into diverse conformations, a structurally invariant outer domain, and, in the CD4-bound state, a bridging sheet minidomain. These studies, however, provide only hints as to the flexibility of each state. Here we use amide hydrogen/deuterium exchange coupled to mass spectrometry to provide quantifications of local conformational stability for HIV-1 gp120 in unliganded and CD4-bound states. On average, unliganded core gp120 displayed >10,000-fold slower exchange of backbone-amide hydrogens than a theoretically unstructured protein of the same composition, with binding by CD4 reducing the rate of gp120 amide exchange a further 10-fold. For the structurally constant CD4, alterations in exchange correlated well with alterations in binding surface ( P value = 0.0004). For the structurally variable gp120, however, reductions in flexibility extended outside the binding surface, and regions of expected high structural diversity (inner domain/bridging sheet) displayed roughly 20-fold more rapid exchange in the unliganded state than regions of low diversity (outer domain). Thus, despite an extraordinary reduction in entropy, neither unliganded gp120 nor free CD4 was substantially unstructured, suggesting that most of the diverse conformations that make up the gp120 unliganded state are reasonably ordered. The results provide a framework for understanding how local conformational stability influences entropic change, conformational diversity, and structural rearrangements in the gp120-CD4 binding reaction.

At least 119 mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis by an unidentified toxic gain of function. We compared the dynamic properties of 13 as-isolated, partially metallated, SOD1 variant enzymes using hydrogen-deuterium exchange. We identified a shared property of these familial amyotrophic lateral sclerosis-related SOD1 variants, namely structural and dynamic change affecting the electrostatic loop (loop VII) of SOD1. Furthermore, SOD1 variants that have severely compromised metal binding affinities demonstrated additional structural and dynamic changes to the zinc-binding loop (loop IV) of SOD1. Although the biological consequences of increased loop VII mobility are not fully understood, this common property is consistent with the hypotheses that SOD1 mutations exert toxicity via aggregation or aberrant association with other cellular constituents.

The ΔF508 mutation in nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the predominant cause of cystic fibrosis. Previous biophysical studies on human F508 and ΔF508 domains showed only local structural changes restricted to residues 509–511 and only minor differences in folding rate and stability. These results were remarkable because ΔF508 was widely assumed to perturb domain folding based on the fact that it prevents trafficking of CFTR out of the endoplasmic reticulum. However, the previously reported crystal structures did not come from matched F508 and ΔF508 constructs, and the ΔF508 structure contained additional mutations that were required to obtain sufficient protein solubility. In this article, we present additional biophysical studies of NBD1 designed to address these ambiguities. Mass spectral measurements of backbone amide 1H/2H exchange rates in matched F508 and ΔF508 constructs reveal that ΔF508 increases backbone dynamics at residues 509–511 and the adjacent protein segments but not elsewhere in NBD1. These measurements also confirm a high level of flexibility in the protein segments exhibiting variable conformations in the crystal structures. We additionally present crystal structures of a broader set of human NBD1 constructs, including one harboring the native F508 residue and others harboring the ΔF508 mutation in the presence of fewer and different solubilizing mutations. The only consistent conformational difference is observed at residues 509–511. The side chain of residue V510 in this loop is mostly buried in all non-ΔF508 structures but completely solvent exposed in all ΔF508 structures. These results reinforce the importance of the perturbation ΔF508 causes in the surface topography of NBD1 in a region likely to mediate contact with the transmembrane domains of CFTR. However, they also suggest that increased exposure of the 509–511 loop and increased dynamics in its vicinity could promote aggregation in vitro and aberrant intermolecular interactions that impede trafficking in vivo.