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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.
Dimattia MA, Watts NR, Stahl SJ, Grimes JM, Steven AC, Stuart DI, Wingfield PT. Antigenic Switching of Hepatitis B Virus by Alternative Dimerization of the Capsid Protein. Structure. 2012 Dec 5. pii: S0969-2126(12)00414-5. doi: 10.1016/j.str.2012.10.017.
Nemecek D, Qiao J, Mindich L, Steven AC, Heymann JB. Packaging accessory protein P7 and polymerase P2 have mutually occluding binding sites inside the bacteriophage 6 procapsid. J Virol. 2012 Nov;86(21):11616-24. doi: 10.1128/JVI.01347-12.
Cardone G, Brecher M, Fontana J, Winkler DC, Butan C, White JM, Steven AC. Visualization of the two-step fusion process of the retrovirus avian sarcoma/leukosis virus by cryo-electron tomography. J Virol. 2012 Nov;86(22):12129-37. doi: 10.1128/JVI.01880-12.
Wu W, Chen Z, Cheng N, Watts NR, Stahl SJ, Farci P, Purcell RH, Wingfield PT, Steven AC. Specificity of an anti-capsid antibody associated with Hepatitis B Virus-related acute liver failure. J Struct Biol. 2012 Oct 16. pii: S1047-8477(12)00272-9. doi: 10.1016/j.jsb.2012.10.004
Cardone G, Newcomb WW, Cheng N, Wingfield PT, Trus BL, Brown JC, Steven AC. The UL36 tegument protein of herpes simplex virus 1 has a composite binding site at the capsid vertices. J Virol. 2012 Apr;86(8):4058-64. doi: 10.1128/JVI.00012-12.
Noinaj N, Easley NC, Oke M, Mizuno N, Gumbart J, Boura E, Steere AN, Zak O, Aisen P, Tajkhorshid E, Evans RW, Gorringe AR, Mason AB, Steven AC, Buchanan SK. Structural basis for iron piracy by pathogenic Neisseria. Nature. 2012 Feb 12;483(7387):53-8. doi: 10.1038/nature10823.See extended list of publications
Reviewed December 21, 2012