Architecture of a dsDNA Viral Capsid in Complex with Its Maturation Protease

Veesler, David; Khayat, Reza; Krishnamurthy, Srinath; Snijder, Joost; Huang, Rick; Heck, Albert J. R.; Anand, Ganesh S.; Johnson, John E.

doi:10.1016/j.str.2013.11.007

Cited by 37 publications

(62 citation statements)

References 29 publications

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“…By massively increasing its interest in protein therapeutics within these last years, the biopharmaceutical industry became particularly interested in improving and developing spatially resolved analytical techniques like HDX-MS for its needs in both research and quality control. (14,15) Since so far HDX-MS was mostly implemented on monomeric proteins (16)(17)(18)(19)(20)(21)(22) and only a few studies have been conducted on homo-oligomeric proteins (23)(24)(25)(26)(27) or protein complexes, (28)(29)(30)(31) efforts have to be continued for extracting the dynamic determinants of complexes.…”

Section: Introductionmentioning

confidence: 99%

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Mechanistic Details of Activation of Nucleoside Diphosphate Kinases by Oligomerization

2017

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Most oligomeric proteins become active only after assembly, but why oligomerization is required to support function is not well understood. Here, we address this question using the WT and a destabilized mutant (D93N) of the hexameric nucleoside diphosphate kinase from the pathogen Mycobacterium tuberculosis (Mt-NDPK). The conformational dynamics and oligomeric states of each were analyzed during unfolding/folding by Hydrogen/Deuterium exchange mass spectrometry (HDX-MS) at peptide resolution and by additional biochemical techniques. We found that WT and D93N native hexamers present a stable core and a flexible periphery, the latter being more flexible for the destabilized mutant. Stable but inactive species formed during unfolding of D93N and folding of WT were characterized. For the first time, we show that both of these species are native-like dimers, each of its monomers having a major subdomain folded, while a minor subdomain (Kpn/α 0 ) remains unfolded. The Kpn/α 0 subdomain, which belongs to the catalytic site, becomes structured only upon hexamerization, explaining why oligomerization is required for NDPK activity. Further HDX-MS studies are necessary to establish the general activation mechanism for other homo-oligomers.

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Section: Introductionmentioning

confidence: 99%

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Mechanistic Details of Activation of Nucleoside Diphosphate Kinases by Oligomerization

2017

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“…Accompanying the exit of scaffolding proteins during subsequent ATP-driven DNA packaging, the icosahedral shell of the procapsid undergoes dramatic conformational changes and matures into a typically larger and more angular shell of the infectious phage (1)(2)(3)(4)(5)(6). However, structural details, including those of capsid intermediates, are limited to the phage HK97 system (5,(7)(8)(9), for which recombinantly produced procapsid and nonphysiological conversion products were analyzed.…”

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confidence: 99%

Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions

Guo

Liu

Fang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

128

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Many dsDNA viruses first assemble a DNA-free procapsid, using a scaffolding protein-dependent process. The procapsid, then, undergoes dramatic conformational maturation while packaging DNA. For bacteriophage T7 we report the following four single-particle cryo-EM 3D reconstructions and the derived atomic models: procapsid (4.6-Å resolution), an early-stage DNA packaging intermediate (3.5 Å), a later-stage packaging intermediate (6.6 Å), and the final infectious phage (3.6 Å). In the procapsid, the N terminus of the major capsid protein, gp10, has a six-turn helix at the inner surface of the shell, where each skewed hexamer of gp10 interacts with two scaffolding proteins. With the exit of scaffolding proteins during maturation the gp10 N-terminal helix unfolds and swings through the capsid shell to the outer surface. The refolded N-terminal region has a hairpin that forms a novel noncovalent, joint-like, intercapsomeric interaction with a pocket formed during shell expansion. These large conformational changes also result in a new noncovalent, intracapsomeric topological linking. Both interactions further stabilize the capsids by interlocking all pentameric and hexameric capsomeres in both DNA packaging intermediate and phage. Although the final phage shell has nearly identical structure to the shell of the DNA-free intermediate, surprisingly we found that the icosahedral faces of the phage are slightly (∼4 Å) contracted relative to the faces of the intermediate, despite the internal pressure from the densely packaged DNA genome. These structures provide a basis for understanding the capsid maturation process during DNA packaging that is essential for large numbers of dsDNA viruses.bacteriophage T7 maturation | DNA packaging intermediates | noncovalent topological linking | procapsid | single-particle cryo-EM M any dsDNA viruses, including tailed phages and herpes viruses, initially assemble a DNA-free procapsid with assistance of a network of scaffold proteins. Accompanying the exit of scaffolding proteins during subsequent ATP-driven DNA packaging, the icosahedral shell of the procapsid undergoes dramatic conformational changes and matures into a typically larger and more angular shell of the infectious phage (1-6). However, structural details, including those of capsid intermediates, are limited to the phage HK97 system (5, 7-9), for which recombinantly produced procapsid and nonphysiological conversion products were analyzed.The packaging of the 39.937-kbp DNA genome of the shorttail Escherichia coli bacteriophage, T7, is a model for understanding basic principles common to dsDNA tailed phages and herpes viruses. The T7 system is also of interest because it has been used for popular biotechnologies, such as recombinant protein expression (10) and protein display on the capsid surface (11). The T7 capsid contains 415 copies of the major shell protein gp10 (12) that form a T = 7L icosahedral lattice. From lowresolution cryo-EM 3D reconstructions the tertiary topology of gp10 can be divided into four regions: N-...

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“…We therefore used amide hydrogen deuterium exchange mass spectrometry (HDXMS) to provide additional insights regarding the dynamic character of the d domain and capsid protein in Prohead I ¡pro and Prohead I Cpro particles. Here we further discuss the implications of the data reported in Veesler et al 4 and provide a working model to explain the deuterium exchange profiles measured for different regions of the viral coat protein. This analysis has implications not discussed in the original paper.…”

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confidence: 68%

“…In these mapping studies, use of a mutant protease stably maintained the Prohead I Cpro intermediate and was used to map capsid protein-protease interactions, focusing on the capsid protein. 4 A total of 140 molecules of protease per virus particle were used, resulting in a 3-fold molar excess of viral capsid protein (420 capsid proteins/virus particle) to protease. HDXMS of the free viral capsid protein provided a relative dynamic profile of the protein at peptide resolution, wherein certain regions were observed to be more dynamic overall as evidenced by their faster deuterium exchange rates (Fig.…”

Section: E959816-2 Volume 4 Issue 4 Bacteriophagementioning

confidence: 99%

“…3 Here we provide an "addendum" to our previous study of Prohead I ¡pro and Prohead I Cpro where we investigated the structural relationship between the d domain and the packaged protease. 4 We established that the d domain in the absence or presence of protease was found to be largely disordered in EM or crystallographic studies suggesting that it adopted multiple conformations. We therefore used amide hydrogen deuterium exchange mass spectrometry (HDXMS) to provide additional insights regarding the dynamic character of the d domain and capsid protein in Prohead I ¡pro and Prohead I Cpro particles.…”

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confidence: 92%

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Distinguishing direct binding interactions from allosteric effects in the protease–HK97 prohead I δ domain complex by amide H/D exchange mass spectrometry

et al. 2014

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A major question in mapping proteinligand or protein-protein interactions in solution is to distinguish direct-binding interactions from long-range conformational changes at allosteric sites. We describe here the applicability of amide hydrogen deuterium exchange mass spectrometry (HDXMS) in addressing this important question using the bacteriophage HK97 capsid proteins' interactions with their processing protease. HK97 is a lambda-like dsDNA bacteriophage that is ideal for studies of particle assembly and maturation. Its capsid precursor protein is composed of two main regions, the scaffolding protein (d-domain, residues 2-103), and the coat subunit (residues 104-385), which spontaneously forms a mixture of hexamers and pentamers upon association. Activation of the viral protease, which occurs after particle assembly, is initiated by the protease mediated digestion of the scaffolding domains to yield Prohead-2. This irreversible step is obligatory for activation of the virus maturation pathway.Here we provide an "addendum" to our previous study of Prohead I and Prohead I Cpro (a transient complex of Prohead I and the protease) where we investigated the interactions between the d domain and the packaged protease using HDXMS. Our results revealed two sites on the d domain: one set of contiguous peptides that showed decreased exchange at the protease binding site at early time points of deuterium labeling and another separate set of continuous peptides that showed decreased exchange at later time points. Even though this cannot reveal the time scales of molecular processes governing binding and allostery, we believe this differential pattern of exchange across deuteration times can allow spatial distinction between binding sites and long range conformational changes with allosteric implications. This partitioning can be discerned from the lag between noncontiguous regions on a protein showing maximal changes in deuterium exchange and highlights a powerful application of HDXMS in distinguishing direct binding in transient protein-protein interactions from allosteric changes.A major question in mapping proteinligand or protein-protein interactions in solution is to distinguish direct binding and allosteric sites. We describe here the applicability of HDXMS in addressing this important question using the bacteriophage HK97 capsid proteins' interaction with their processing protease. HK97 is a lambda-like dsDNA bacteriophage that is exceptionally amenable for studies of particle assembly and maturation.1 Prohead I ¡pro is the first assembly product when the capsid protein gene (gp5) is expressed in E. coli. It is formed by 420 copies of the subunit arranged on a T D 7l quasi-equivalent surface lattice. HK97 gp5 is a 384 amino acid protein with residues 2-103 (called the d domain) required for proper assembly of the capsid. Expression of the viral protease (gp4) with gp5 results in a transient particle, Prohead ICpro , that undergoes proteolysis of the d domain when the particle is fully assembled,

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Architecture of a dsDNA Viral Capsid in Complex with Its Maturation Protease

Cited by 37 publications

References 29 publications

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Mechanistic Details of Activation of Nucleoside Diphosphate Kinases by Oligomerization

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Mechanistic Details of Activation of Nucleoside Diphosphate Kinases by Oligomerization

Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions

Distinguishing direct binding interactions from allosteric effects in the protease–HK97 prohead I δ domain complex by amide H/D exchange mass spectrometry

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