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| Subject Categories: Structural Biology | Microbiology & Pathogens |
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| The EMBO Journal
(2005) 24, 1352–1363, doi:10.1038/sj.emboj.7600613 Published online 17 March 2005 |
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| Crosslinking renders bacteriophage HK97 capsid maturation irreversible and effects an essential stabilization |
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| Philip D Ross1, Naiqian Cheng2, James F Conway3, Brian A Firek4, Roger W Hendrix4, Robert L Duda4 and Alasdair C Steven2 |
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1 Laboratory of Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, MD, USA 2 Laboratory of Structural Biology Research, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, MD, USA 3 Institut de Biologie Structurale CEA-CNRS-UJF, Grenoble, France 4 Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA To whom correspondence should be addressed Alasdair C Steven, Building 50, Room 1517, 50 South Drive MSC 8025, NIH, Bethesda, MD 20892, USA. Tel.: +1 301 443 7651; Fax: +1 301 496 0132; E-mail: alasdair_steven@nih.gov Received 1 December 2004; Accepted 10 February 2005; Published online 17 March 2005. |
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| Abstract |
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In HK97 capsid maturation, structural change ('expansion') is accompanied by formation of covalent crosslinks, connecting residue K169 in the 'E-loop' of each subunit with N356 on another subunit. We show by complementation experiments with the K169Y mutant, which cannot crosslink, that crosslinking is an essential function. The precursor Prohead-II passes through three expansion intermediate (EI) states en route to the end state, Head-II. We investigated the effects of expansion and crosslinking on stability by differential scanning calorimetry of wild-type and K169Y capsids. After expansion, the denaturation temperature (Tp) of K169Y capsids is slightly reduced, indicating that their thermal stability is not enhanced, but crosslinking effects a major stabilization ( Tp, +11°C). EI-II is the earliest capsid to form crosslinks. Cryo-electron microscopy shows that for both wild-type and K169Y EI-II, most E-loops are in the 'up' position, 30 Å from the nearest N356: thus, crosslinking in EI-II represents capture of mobile E-loops in 'down' positions. At pH 4, most K169Y capsids remain as EI-II, whereas wild-type capsids proceed to EI-III, suggesting that crosslink formation drives maturation by a Brownian ratchet mechanism. |
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| Keywords: Brownian ratchet, conformational change, cryo-electron microscopy, differential scanning calorimetry, virus assembly |
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