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Laboratory of Structural Biology Research
The Laboratory of Structural Biology Research seeks to elucidate structure-function-assembly relationships of macromolecular complexes by cryo-electron microscopy integrated with other approaches. Systems currently under study include viruses, cytoskeletal filaments, energy-dependent proteases, and amyloid filaments.
The structural basis of virus replication has been a long-standing major interest of this laboratory, with particular focus on the roles of conformational changes in regulating two critical steps in the cycle - assembly and maturation of the nucleocapsid; and recognition of susceptible hosts and cell entry. Systems currently under study are: capsid structure and antigenicity of hepatitis B virus; procapsid assembly and maturation of herpes simplex virus and its subsequent acquisition of a tegument (protein compartment between the capsid and the envelope); cell entry of poliovirus, focusing on its interaction with its receptor and the consequent capsid transition that leads to cell penetration and genome release. Our work on double-stranded DNA phages centers on the large-scale conformational changes that accompany capsid maturation, the organization of encapsidated DNA, and biotechnological applications of their dispensable capsid proteins. We also study the internal organization of papillomavirus.
The LSBR has a long-standing commitment to the IATAP - Intramural Targeted Antiviral - program at NIH, which brings expertise existing in the Intramural program to problems bearing on HIV and AIDS. We continue to participate actively in a number of studies related to this program.
Elimination of misfolded and foreign proteins is an essential function of cells, carried out by energy-dependent proteases. Because these enzymes function inside cells, stringent mechanisms must be in place to confine their activity to bona fide targets, sparing endogenous proteins. Generally, they consist of two subcomplexes: an oligomeric peptidase, and an ATP-hydrolysing chaperone that recognizes substrates and presents them for proteolysis. In eukaryotic cells, most such activity is carried out by proteasomes. The relatively simple 2-component proteases of bacteria offer tractable model systems and we have been studying those of E. coli (with M. Maurizi, NCI). Initially we determined their oligomeric structures and found a symmetry mismatch between the heptameric peptidase ClpP, and the hexameric ATPases ClpA and ClpX. The proteasome appears to exhibit a similar mismatch but other bacterial enzymes do not require one. It has transpired that all the ATPases are members of the AAA family for which some dozen-crystal structures are on record. Currently, the primary role for EM is to characterize the mode of interaction of the peptidase with the ATPase in forming intact holoenzymes and their processing of substrate proteins. We have shown that substrates initially bind to distal sites on these barrel-like complexes and are subsequently translocated along an axial pathway into the internal chamber where the active sites reside. We are now pursuing more detailed aspects of this overall process.
We are studying a diverse set of proteins that have in common fibrous/filamentous conformations that are rich in beta-sheet conformations: (i) amyloid filaments of the yeast prion protein, Ure2p; (ii) secreted bacterial proteins typified by the filamentous hemaggutinin of B. pertussis; (iii) viral receptor-recognition proteins, typified by the tail-fibers of bacteriophages. Yeast has several proteins that manifest the phase changes and genetic properties typical of the prion proteins that are associated with certain important neuropathologies. In doing so, they form amyloid filaments similar to those also involved in a wider range of diseases including rheumatoid arthritis. In their "prion" form, they are assembled into amyloid-containing filaments in which the protein is inactive. We are studying the assembly and structure of filaments and the mechanism of inactivation in yeast prionogenesis of Ure2p (with R. Wickner, NIDDK). Our current picture is that the N-terminal prion domain controls filament formation, undergoing a major conformational change on entering the polymeric state: the enzymatic domain appears to be inactivated by steric blocking from its reaction partner in the filament, not by refolding. Amyloid-like conformations are employed in the native folds of phage tail-fibers and secreted bacterial proteins and we are investigating these molecules by electron microscopy, molecular modeling and related approaches.
Macromolecular Complexes in Skin and Muscle
We study several complexes that form integral components of skin (specifically, the epidermis) or are related to muscle filament function. The cornified cell envelope is a covalently cross-linked sheet of protein that forms at the surface of terminally differentiated keratinocytes and is thought to play an important role in specifying the permeability properties of the epidermis. Based on EM and other observations, we have developed the concept of the CE as a composite biomaterial, consisting of a "filament" component and a "matrix" component: the biomechanical properties of the CE are "tuned" to the requirements of each cornifying epithelium by appropriate adjustment of the nature and relative amounts of both components. Backup systems are available to substitute when major components are eliminated in knockout mice. We have elucidated the pathogenic mechanism whereby the major CE component is subverted in genetic skin diseases like Vohwinkel's syndrome (with D. Roop, Baylor). Another composite biomaterial in the epidermis is the keratin intermediate filament matrix that constitutes the cornified cytoplasm. We have pursued a long-term program of studying the structures of IF from numerous sources, most recently, native keratin filaments from hair follicles. Actin-stimulated ATPase activity of myosin is the basic mechanism underlying force generation in muscular contraction. We are study the structural basis of this process in the analagous system of acto-myosin I (a monomeric myosin), with particular emphasis on regulation by phosphorylation and on the disposition of the non-motor domains of this myosin.
Although most projects undertaken in the LSBR are interdisciplinary in character, they generally include EM and image processing experiments as major components. We have a longstanding practice of developing and applying novel methods in both areas including, in particular, the PIC image processing system; programs for processing data to facilitate high resolution reconstructions from cryo-EM data; and specialized algorithms for symmetry detection and other tasks. In 1997, the LSBR was one of the first laboratories to calculate three-dimensional density maps of "single particles" to resolutions higher than 10 Α, revealing alpha-helical sub-structure. This effort is ongoing: we are currently implementing an innovative EM equipped with a field-emission gun and compatibility to operate at liquid-helium temperature; developing additional software; and we are making a start in electron tomography.
Pérez-Berná AJ, Marion S, Chichón FJ, Fernández JJ, Winkler DC, Carrascosa JL, Steven AC, iber A, San Martín C. Distribution of DNA-condensing protein complexes in the adenovirus core. Nucleic Acids Res. 2015 Mar 27. pii: gkv187. [Epub ahead of print]
Cardone G, Duda RL, Cheng N, You L, Conway JF, Hendrix RW, Steven AC. Metastable intermediates as stepping stones on the maturation pathways of viral capsids. MBio. 2014 Nov 11;5(6):e02067. doi: 10.1128/mBio.02067-14.
McHugh CA, Fontana J, Nemecek D, Cheng N, Aksyuk AA, Heymann JB, Winkler DC, Lam AS, Wall JS, Steven AC, Hoiczyk E. A virus capsid-like nanocompartment that stores iron and protects bacteria from oxidative stress. EMBO J. 2014 Sep 1;33(17):1896-911. doi: 10.15252/embj.201488566. Epub 2014 Jul 14.
Liu Y, Jesus AA, Marrero B, Yang D, Ramsey SE, Montealegre Sanchez GA, Tenbrock K, Wittkowski H, Jones OY, Kuehn HS, Lee CC, DiMattia MA, Cowen EW, Gonzalez B, Palmer I, DiGiovanna JJ, Biancotto A, Kim H, Tsai WL, Trier AM, Huang Y, Stone DL, Hill S, Kim HJ, St Hilaire C, Gurprasad S, Plass N, Chapelle D, Horkayne-Szakaly I, Foell D, Barysenka A, Candotti F, Holland SM, Hughes JD, Mehmet H, Issekutz AC, Raffeld M, McElwee J, Fontana JR, Minniti CP, Moir S, Kastner DL, Gadina M, Steven AC, Wingfield PT, Brooks SR, Rosenzweig SD, Fleisher TA, Deng Z, Boehm M, Paller AS, Goldbach-Mansky R. Activated STING in a Vascular and Pulmonary Syndrome. The New England Journal of Medicine 2014 Aug 7;371(6):507-18. doi: 10.1056/NEJMoa1312625. Epub 2014 Jul 16.
Cardone G, Moyer AL, Cheng N, Thompson CD, Dvoretzky I, Lowy DR, Schiller JT, Steven AC, Buck CB, Trus BL. Maturation of the human papillomavirus 16 capsid. MBio. 2014 Aug 5;5(4):e01104-14. doi: 10.1128/mBio.01104-14.
Zhuang X, Stahl SJ, Watts NR, DiMattia MA, Steven AC, Wingfield PT. A cell-penetrating antibody fragment against HIV-1 Rev has high antiviral activity: characterization of the paratope. J Biol Chem. 2014 Jul 18;289(29):20222-33. doi: 10.1074/jbc.M114.581090. Epub 2014 May 30.
Hickman AB, Ewis HE, Li X, Knapp JA, Laver T, Doss AL, Tolun G, Steven AC, Grishaev A, Bax A, Atkinson PW, Craig NL, Dyda F. Structural basis of hAT transposon end recognition by Hermes, an octameric DNA transposase from Musca domestica. Cell. 2014 Jul 17;158(2):353-67. doi: 10.1016/j.cell.2014.05.037.
Bereszczak JZ, Watts NR, Wingfield PT, Steven AC, Heck AJ. Assessment of differences in the conformational flexibility of hepatitis B virus core-antigen and e-antigen by hydrogen deuterium exchange-mass spectrometry. Protein Sci. 2014 Jul;23(7):884-96. doi: 10.1002/pro.2470. Epub 2014 Apr 17.
Huang RK, Baxa U, Aldrian G, Ahmed AB, Wall JS, Mizuno N, Antzutkin O, Steven AC, Kajava AV. Conformational switching in PolyGln amyloid fibrils resulting from a single amino acid insertion. Biophys J. 2014 May 20;106(10):2134-42. doi: 10.1016/j.bpj.2014.03.047.
Cairns TM, Fontana J, Huang ZY, Whitbeck JC, Atanasiu D, Rao S, Shelly SS, Lou H, Ponce de Leon M, Steven AC, Eisenberg RJ, Cohen GH. Mechanism of neutralization of herpes simplex virus by antibodies directed at the fusion domain of glycoprotein B. J Virol. 2014 Mar;88(5):2677-89. doi: 10.1128/JVI.03200-13. Epub 2013 Dec 18.See extended list of publications
Reviewed December 21, 2012