Meytal Landau

ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures

Key amino acid positions that are important for maintaining the 3D structure of a protein and/or its function(s), e.g. catalytic activity, binding to ligand, DNA or other proteins, are often under strong evolutionary constraints. Thus, the biological importance of a residue often correlates with its level of evolutionary conservation within the protein family. ConSurf () is a web-based tool that automatically calculates evolutionary conservation scores and maps them on protein structures via a user-friendly interface. Structurally and functionally important regions in the protein typically appear as patches of evolutionarily conserved residues that are spatially close to each other. We present here version 3.0 of ConSurf. This new version includes an empirical Bayesian method for scoring conservation, which is more accurate than the maximum-likelihood method that was used in the earlier release. Various additional steps in the calculation can now be controlled by a number of advanced options, thus further improving the accuracy of the calculation. Moreover, ConSurf version 3.0 also includes a measure of confidence for the inferred amino acid conservation scores.

Atomic View of a Toxic Amyloid Small Oligomer

A Toxic Barrel Many studies have suggested that oligomers are an important toxic species in amyloid diseases such as Alzheimers disease. In an effort to better define these oligomers, Laganowsky et al. (p. 1228) identified a segment of the fibril-forming protein αB crystalline (ABC) that forms both amyloid fibrils and a relatively stable oligomer. ABC oligomers were toxic in a cell viability assay and were recognized by an amyloid-oligomer–specific antibody. A crystal structure of the oligomers showed that six peptides formed an antiparallel barrel termed a cylindrin. Amyloid oligomers are likely to be structurally polymorphic, but cylindrin-like assemblies offer a model for these elusive structures. Cylindrin from the amyloid-forming protein αB crystallin represents an amyloid oligomer. Amyloid diseases, including Alzheimer’s, Parkinson’s, and the prion conditions, are each associated with a particular protein in fibrillar form. These amyloid fibrils were long suspected to be the disease agents, but evidence suggests that smaller, often transient and polymorphic oligomers are the toxic entities. Here, we identify a segment of the amyloid-forming protein αB crystallin, which forms an oligomeric complex exhibiting properties of other amyloid oligomers: β-sheet–rich structure, cytotoxicity, and recognition by an oligomer-specific antibody. The x-ray–derived atomic structure of the oligomer reveals a cylindrical barrel, formed from six antiparallel protein strands, that we term a cylindrin. The cylindrin structure is compatible with a sequence segment from the β-amyloid protein of Alzheimer’s disease. Cylindrins offer models for the hitherto elusive structures of amyloid oligomers.

Molecular Basis for Amyloid-{Beta} Polymorphism.

Amyloid-beta (Aβ) aggregates are the main constituent of senile plaques, the histological hallmark of Alzheimer’s disease. Aβ molecules form β-sheet containing structures that assemble into a variety of polymorphic oligomers, protofibers, and fibers that exhibit a range of lifetimes and cellular toxicities. This polymorphic nature of Aβ has frustrated its biophysical characterization, its structural determination, and our understanding of its pathological mechanism. To elucidate Aβ polymorphism in atomic detail, we determined eight new microcrystal structures of fiber-forming segments of Aβ. These structures, all of short, self-complementing pairs of β-sheets termed steric zippers, reveal a variety of modes of self-association of Aβ. Combining these atomic structures with previous NMR studies allows us to propose several fiber models, offering molecular models for some of the repertoire of polydisperse structures accessible to Aβ. These structures and molecular models contribute fundamental information for understanding Aβ polymorphic nature and pathogenesis.

Crystal structures of truncated alphaA and alphaB crystallins reveal structural mechanisms of polydispersity important for eye lens function.

Small heat shock proteins alphaA and alphaB crystallin form highly polydisperse oligomers that frustrate protein aggregation, crystallization, and amyloid formation. Here, we present the crystal structures of truncated forms of bovine alphaA crystallin (AAC59–163) and human alphaB crystallin (ABC68–162), both containing the C‐terminal extension that functions in chaperone action and oligomeric assembly. In both structures, the C‐terminal extensions swap into neighboring molecules, creating runaway domain swaps. This interface, termed DS, enables crystallin polydispersity because the C‐terminal extension is palindromic and thereby allows the formation of equivalent residue interactions in both directions. That is, we observe that the extension binds in opposite directions at the DS interfaces of AAC59–163 and ABC68–162. A second dimeric interface, termed AP, also enables polydispersity by forming an antiparallel beta sheet with three distinct registration shifts. These two polymorphic interfaces enforce polydispersity of alpha crystallin. This evolved polydispersity suggests molecular mechanisms for chaperone action and for prevention of crystallization, both necessary for transparency of eye lenses.

Towards a pharmacophore for amyloid.

Diagnosing and treating Alzheimers and other diseases associated with amyloid fibers remains a great challenge despite intensive research. To aid in this effort, we present atomic structures of fiber-forming segments of proteins involved in Alzheimers disease in complex with small molecule binders, determined by X-ray microcrystallography. The fiber-like complexes consist of pairs of β-sheets, with small molecules binding between the sheets, roughly parallel to the fiber axis. The structures suggest that apolar molecules drift along the fiber, consistent with the observation of nonspecific binding to a variety of amyloid proteins. In contrast, negatively charged orange-G binds specifically to lysine side chains of adjacent sheets. These structures provide molecular frameworks for the design of diagnostics and drugs for protein aggregation diseases.

Model Structure of the Na+/H+ Exchanger 1 (NHE1) FUNCTIONAL AND CLINICAL IMPLICATIONS

Eukaryotic Na+/H+ exchangers are transmembrane proteins that are vital for cellular homeostasis and play key roles in pathological conditions such as cancer and heart diseases. Using the crystal structure of the Na+/H+ antiporter from Escherichia coli (EcNhaA) as a template, we predicted the three-dimensional structure of human Na+/H+ exchanger 1 (NHE1). Modeling was particularly challenging because of the extremely low sequence identity between these proteins, but the model structure is supported by evolutionary conservation analysis and empirical data. It also revealed the location of the binding site of NHE inhibitors; which we validated by conducting mutagenesis studies with EcNhaA and its specific inhibitor 2-aminoperimidine. The model structure features a cluster of titratable residues that are evolutionarily conserved and are located in a conserved region in the center of the membrane; we suggest that they are involved in the cation binding and translocation. We also suggest a hypothetical alternating-access mechanism that involves conformational changes.

Structure-based discovery of fiber-binding compounds that reduce the cytotoxicity of amyloid beta

Amyloid protein aggregates are associated with dozens of devastating diseases including Alzheimer’s, Parkinson’s, ALS, and diabetes type 2. While structure-based discovery of compounds has been effective in combating numerous infectious and metabolic diseases, ignorance of amyloid structure has hindered similar approaches to amyloid disease. Here we show that knowledge of the atomic structure of one of the adhesive, steric-zipper segments of the amyloid-beta (Aβ) protein of Alzheimer’s disease, when coupled with computational methods, identifies eight diverse but mainly flat compounds and three compound derivatives that reduce Aβ cytotoxicity against mammalian cells by up to 90%. Although these compounds bind to Aβ fibers, they do not reduce fiber formation of Aβ. Structure-activity relationship studies of the fiber-binding compounds and their derivatives suggest that compound binding increases fiber stability and decreases fiber toxicity, perhaps by shifting the equilibrium of Aβ from oligomers to fibers. DOI: http://dx.doi.org/10.7554/eLife.00857.001

Functional evaluation of autism-associated mutations in NHE9

Summary NHE9 (SLC9A9) is an endosomal cation/proton antiporter with orthologs in yeast and bacteria. Rare, missense substitutions in NHE9 are genetically linked with autism, but have not been functionally evaluated. Here we use evolutionary conservation analysis to build a model-structure of NHE9 based on the crystal structure of bacterial NhaA and use it to screen autism-associated variants in the human population first by phenotype complementation in yeast, followed by functional analysis in primary cortical astrocytes from mouse. NHE9-GFP localizes to recycling endosomes where it significantly alkalinizes luminal pH, elevates uptake of transferrin and the neurotransmitter glutamate, and stabilizes surface expression of transferrin receptor and GLAST transporter. In contrast, autism associated variants L236S, S438P and V176I lack function in astrocytes. Thus, we establish a neurobiological cell model of a candidate gene in autism. Loss of function mutations in NHE9 may contribute to autistic phenotype by modulating synaptic membrane protein expression and neurotransmitter clearance.

Specific Cysteines in β3 Are Involved in Disulfide Bond Exchange-dependent and -independent Activation of αIIbβ3

Disulfide bond exchange among cysteine residues in epidermal growth factor (EGF)-like domains of β3 was suggested to be involved in activation of αIIbβ3. To investigate the role of specific β3 cysteines in αIIbβ3 expression and activation, we expressed in baby hamster kidney cells normal αIIb with normal β3 or β3 with single or double cysteine substitutions of nine disulfide bonds in EGF-3, EGF-4, and β-tail domains and assessed αIIbβ3 surface expression and activation state by flow cytometry using P2 or PAC-1 antibodies, respectively. Most mutants displayed reduced surface expression of αIIbβ3. Disruptions of disulfide bonds in EGF-3 yielded constitutively active αIIbβ3, implying that these bonds stabilize the inactive αIIbβ3 conformer. Mutants of the Cys-567–Cys-581 bond in EGF-4 were inactive even after exposure to αIIbβ3-activating antibodies, indicating that this bond is necessary for activating αIIbβ3. Disrupting Cys-560–Cys-583 in the EGF-3/EGF-4 or Cys-608–Cys-655 in β-tail domain resulted in αIIbβ3 activation only when Cys-560 or Cys-655 of each pair was mutated but not when their partners (Cys-583, Cys-608) or both cysteines were mutated, suggesting that free sulfhydryls of Cys-583 and Cys-608 participate in αIIbβ3 activation by a disulfide bond exchange-dependent mechanism. The free sulfhydryl blocker dithiobisnitrobenzoic acid inhibited 70% of anti-LIBS6 antibody-induced activation of wild-type αIIbβ3 and had a smaller effect on mutants, implicating disulfide bond exchange-dependent and -independent mechanisms in αIIbβ3 activation. These data suggest that different disulfide bonds in β3 EGF and β-tail domains play variable structural and regulatory roles in αIIbβ3.

The cytotoxic Staphylococcus aureus PSMα3 reveals a cross-α amyloid-like fibril.

Whats in a fold? Bacterially secreted peptides known as PSMs (phenol-soluble modulins) stimulate inflammatory responses, lyse human cells, and contribute to biofilm structuring. PSMα3 is a virulent 22-residue amyloid peptide secreted by Staphylococcus aureus. Tayeb-Fligelman et al. present a high-resolution structure encompassing the full length of the amyloids sequence. This structure reveals an unexpected departure from the common amyloid cross-β folded architecture. Instead, PSMα3 forms amphipathic α-helices that are folded to stack perpendicular to the fibril axis into sheets. This unusual cross-α structure was important for fibril toxicity. Science, this issue p. 831 Fibrillation-dependent cytotoxicity of PSMα3 functional amyloid is encoded by an unusual cross-α peptide architecture. Amyloids are ordered protein aggregates, found in all kingdoms of life, and are involved in aggregation diseases as well as in physiological activities. In microbes, functional amyloids are often key virulence determinants, yet the structural basis for their activity remains elusive. We determined the fibril structure and function of the highly toxic, 22-residue phenol-soluble modulin α3 (PSMα3) peptide secreted by Staphylococcus aureus. PSMα3 formed elongated fibrils that shared the morphological and tinctorial characteristics of canonical cross-β eukaryotic amyloids. However, the crystal structure of full-length PSMα3, solved de novo at 1.45 angstrom resolution, revealed a distinctive “cross-α” amyloid-like architecture, in which amphipathic α helices stacked perpendicular to the fibril axis into tight self-associating sheets. The cross-α fibrillation of PSMα3 facilitated cytotoxicity, suggesting that this assembly mode underlies function in S. aureus.