Recently in Science Highlights Category

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Central challenges in the design of large and dynamic macromolecular assemblies for synthetic biology lie in developing effective methods for testing design strategies and their outcomes, including comprehensive assessments of solution behavior. The authors of this paper created and validated an advanced design of a 600-kDa protein homododecamer that self-assembles into a symmetric tetrahedral cage. The monomeric unit is composed of a trimerizing apex-forming domain genetically linked to an edge-forming dimerizing domain. Enhancing the crystallographic results, high-throughput small-angle x-ray scattering (SAXS) comprehensively contrasted our modifications under diverse solution conditions. To generate a phase diagram associating structure and assembly, we developed force plots that measure dissimilarity among multiple SAXS data sets. These new tools, which provided effective feedback on experimental constructs relative to design, have general applicability in analyzing the solution behavior of heterogeneous nanosystems and have been made available as a web-based application. Specifically, our results probed the influence of solution conditions and symmetry on stability and structural adaptability, identifying the dimeric interface as the weak point in the assembly. Force plots comparing SAXS data sets further reveal more complex and controllable behavior in solution than captured by our crystal structures. These methods for objectively and comprehensively comparing SAXS profiles for systems critically affected by solvent conditions and structural heterogeneity provide an enabling technology for advancing the design and bioengineering of nanoscale biological materials.


Yen-Ting Lai1, Greg L. Hura, Kevin N. Dyer, Henry Y. H. Tang, John A. Tainer and Todd O. Yeates Designing and defining dynamic protein cage nanoassemblies in solution 14 Dec 2016:Vol. 2, no. 12

In this paper, the authors show that CRY1, a protein coding gene that activates circadian gene expression and metabolic states and circadian oscillators, binds directly to the PAS domain core of CLOCK:BMAL1. Precise control of CLOCK:BMAL1 activity by coactivators and repressors establishes the ~24 hr periodicity of gene expression. Integrative modeling and solution X-ray scattering studies (conducted at the SIBYLS beamline 12.3.1) irrefutably position a key loop of the CLOCK PAS-B domain in the secondary pocket of CRY1, analogous to the antenna chromophore-binding pocket of photolyase. This study is significant for understanding the clock mechanism as fundamental for the development and application of therapies for circadian-related disorders.

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SAXS profile of CRY1:CLOCK:BMAL1 repressive complex.

(A) Scattering traces of CRY1:CLOCK:BMAL1 ternary complex (CCB) at different con- centrations are shown. These scattering plots were merged to generate the dataset as the input for FoXSDock. (B) Guinier analysis of CCB shows little or no aggregation of sample. SAXS-calculated molecular weight of the ternary complex is 113 kDa. (C) Kratky plot shows the CCB complex indicates a folded mass with an elongated shape. (D) PDB of FoXSDock HADDOCK driven model that is among the top 20 nearly degenerate docking structures, χ = 2.74.


Michael AK, Fribourgh L, Chelliah Y, Sandate C, Hura GL, Schneidman-Duhovny, Tripathi SM, Takahashi JS, Partch CL “Formation of a repressive complex in the mammalian circadian clock is mediated by the secondary pocket of CRY1” PNAS 2017 Jan 31, doi:10.1073/pnas.1615310114

Apoptosis-inducing factor (AIF) is critical for mitochondrial respiratory complex biogenesis and for mediating necroptotic parthanatos; these functions are seemingly regulated by enigmatic allosteric switching driven by NADH charge-transfer complex (CTC) formation. In this paper the authors define molecular pathways linking AIF’s active site to allosteric switching regions by characterizing dimer-permissive mutants using small-angle X-ray scattering (SAXS) and crystallography and by probing AIF-CTC communication networks using molecular dynamics simulations. Collective results identify two pathways propagating allostery from the CTC active site: (1) active-site H454 links to S480 of AIF’s central β-strand to modulate a hydrophobic border at the dimerization interface, and (2) an interaction network links AIF’s FAD cofactor, central β-strand, and Cβ-clasp whereby R529 reorientation initiates C-loop release during CTC formation. This knowledge of AIF allostery and its flavoswitch mechanism provides a foundation for biologically understanding and biomedically controlling its participation in mitochondrial homeostasis and cell death.

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Brosey CA, Ho C, Long WZ, Singh S, Burnett K, Hura GL, Nix JC, Bowman GR, Ellenberger T, Tainer JA. “Defining NADH-Driven Allostery Regulating Apoptosis-Inducing Factor.” Structure 2016 Dec 06 ;24(12)

Tip link filaments convey force and gate inner-ear hair-cell transduction channels to mediate perception of sound and head movements. Cadherin-23 and protocadherin-15 form tip links through a calcium-dependent interaction of their extracellular domains made of multiple extracellular cadherin (EC) repeats. These repeats are structurally similar, but not identical in sequence, often featuring linkers with conserved calcium-binding sites that confer mechanical strength to them. In a paper recently published in Nature Communications the Sotomayor lab reports the X-ray crystal structures of human protocadherin-15 EC8-EC10 and mouse EC9-EC10, which show an EC8-9 canonical-like calcium-binding linker, and an EC9-10 calcium-free linker that alters the linear arrangement of EC repeats. Molecular dynamics simulations and small-angle X-ray scattering experiments support this non-linear conformation. Simulations also suggest that unbending of EC9-10 confers some elasticity to otherwise rigid tip links. The new structure provides a first view of protocadherin-15’s non- canonical EC linkers and suggests how they may function in inner-ear mechanotransduction, with implications for other cadherins.

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Araya-Secchi R, Neel BL, Sotomayor M. “An elastic element in the protocadherin-15 tip link of the inner ear.” Nat Commun 2016 Nov 18 ;7

The facilities and staff at the SYBILS beamline contributed to this breakthrough study exploring the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain.

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Naturally occurring proteins—chains of amino acids that fold into functional, three-dimensional shapes—are believed to represent just a small fraction of the universe of all possible permutations of amino-acid sequences and folds. How can we begin to systematically sift through those permutations to find and engineer from scratch (de novo) proteins with the characteristics desired for medical, environmental, and industrial purposes? To address this question, a team led by researchers from the Institute for Protein Design at the University of Washington have published a landmark study that used both protein crystallography and small-angle x-ray scattering (SAXS) at the ALS to validate the computationally designed structures of novel proteins with repeated motifs. The results show that the protein-folding universe is far larger than realized, opening up a wide array of new possibilities for biomolecular engineering.

Brunette TJ, Parmeggiani F, Huang PS, Bhabha G, Ekiert DC, Tsutakawa SE, Hura GL, Tainer JA, Baker D. “Exploring the repeat protein universe through computational protein design.” Nature 2015 Dec 24 ;528(7583)link

The facilities and staff at the SIBYLS beamline contributed to this recent structural study of a human antibody that is able to neutralize Marburg and Ebola viruses.

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The filoviruses, including Marburg and Ebola, express a single glycoprotein on their surface, termed GP, which is responsible for attachment and entry of target cells. Filovirus GPs differ by up to 70% in protein sequence, and no antibodies are yet described that cross-react among them. Here, we present the 3.6 Å crystal structure of Marburg virus GP in complex with a cross-reactive antibody from a human survivor, and a lower resolution structure of the antibody bound to Ebola virus GP. The antibody, MR78, recognizes a GP1 epitope conserved across the filovirus family, which likely represents the binding site of their NPC1 receptor. Indeed, MR78 blocks binding of the essential NPC1 domain C. These structures and additional small-angle X-ray scattering of mucin-containing MARV and EBOV GPs suggest why such antibodies were not previously elicited in studies of Ebola virus, and provide critical templates for development of immunotherapeutics and inhibitors of entry.

Hashiguchi T, Fusco ML, Bornholdt ZA, Lee JE, Flyak AI, Matsuoka R, Kohda D, Yanagi Y, Hammel M, Crowe JE, Saphire EO. “Structural basis for marburg virus neutralization by a cross-reactive human antibody.” Cell 2015 Feb 26 ;160(5):904-12 link

SAXS and MX data collected at SIBYLS enabled insights into protein flexibility in one of the proteins involved in Lou Gerigh’s disease. The results were recently published in PNAS and have been highlighted by Today At Berkeley Lab and and Daily Cal.

Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misassembly and aggregation in cells affected by the motor neuron disease ALS. However, the mechanistic relationship between superoxide dismutase 1 (SOD1) mutations and human disease is controversial, with many hypotheses postulated for the propensity of specific SOD mutants to cause ALS. Here, we experimentally identify distinguishing attributes of ALS mutant SOD proteins that correlate with clinical severity by applying solution biophysical techniques to six ALS mutants at human SOD hotspot glycine 93. A small-angle X-ray scattering (SAXS) assay and other structural methods assessed aggregation propensity by defining the size and shape of fibrillar SOD aggregates after mild biochemical perturbations. Inductively coupled plasma MS quantified metal ion binding stoichiometry, and pulsed dipolar ESR spectroscopy evaluated the Cu2+ binding site and defined cross-dimer copper-copper distance distributions. Importantly, we find that copper deficiency in these mutants promotes aggregation in a manner strikingly consistent with their clinical severities. G93 mutants seem to properly incorporate metal ions under physiological conditions when assisted by the cop- per chaperone but release copper under destabilizing conditions more readily than the WT enzyme. Altered intradimer flexibility in ALS mutants may cause differential metal retention and promote distinct aggregation trends observed for mutant proteins in vitro and in ALS patients. Combined biophysical and structural results test and link copper retention to the framework destabilization hypothesis as a unifying general mechanism for both SOD aggregation and ALS disease progression, with implications for disease severity and therapeutic intervention strategies.

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Pratt AJ, Shin DS, Merz GE, Rambo RP, Lancaster WA, Dyer KN, Borbat PP, Poole FL, Adams MW, Freed JH, Crane BR, Tainer JA, Getzoff ED. “Aggregation propensities of superoxide dismutase G93 hotspot mutants mirror ALS clinical phenotypes.” Proc. Natl. Acad. Sci. U.S.A. 2014 Oct 14 link

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