Structural Analysis of Activated SgrAI–DNA Oligomers Using Ion Mobility Mass Spectrometry

Ma, Xin; Shah, Santosh; Zhou, Mowei; Park, Chad K.; Wysocki, Vicki H.; Horton, Nancy C.

doi:10.1021/bi3013214

Cited by 22 publications

(29 citation statements)

References 66 publications

(206 reference statements)

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“…1C). This analysis shows that the low-activity SgrAI DBD that was previously characterized crystallographically (Dunten et al, 2008; Little et al, 2011; Park et al, 2010a) can oligomerize in a run-on fashion in the presence of activating DNA, supporting a current hypothesis from mass spectrometry data (Ma et al, 2013). …”

Section: Resultssupporting

confidence: 77%

Allosteric Regulation of DNA Cleavage and Sequence-Specificity through Run-On Oligomerization

et al. 2013

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SgrAI is a sequence specific DNA endonuclease that functions through an unusual enzymatic mechanism that is allosterically activated 200-500 fold by effector DNA, with a concomitant expansion of its DNA sequence specificity. Using single-particle transmission electron microscopy to reconstruct distinct populations of SgrAI oligomers, we show that, in the presence of allosteric, activating DNA, the enzyme forms regular, repeating helical structures that are characterized by the addition of DNA-binding dimeric SgrAI subunits in a run-on manner. We also present the structure of oligomeric SgrAI at 8.6 Å resolution, demonstrating a novel conformational state of SgrAI in its activated form. Activated and oligomeric SgrAI displays key protein-protein interactions near the helix axis between its N-termini, as well as allosteric protein-DNA interactions that are required for enzymatic activation. The hybrid approach reveals an unusual mechanism of enzyme activation that explains SgrAI’s oligomerization and allosteric behavior.

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Section: Resultssupporting

confidence: 77%

Allosteric Regulation of DNA Cleavage and Sequence-Specificity through Run-On Oligomerization

et al. 2013

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“…The exception is nucleotide CCS values, which, aside for the 2009 study from McLean and coworkers, 68 have been published in small numbers spread across several studies and years, and currently comprise about 1% all CCS values reported. 153,199–209 This observation is reflected in the fact that while many of these IM-based biomolecular studies have coincided strongly with developments in MS-based lipidomics, glycomics and metabolomics, the role of mass spectrometry related techniques in genomics research is, and has always been, relatively small.…”

Section: Ccs Coverage Over Timementioning

confidence: 99%

Ion Mobility Collision Cross Section Compendium

2016

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In this review, we focus on an important aspect of ion mobility (IM) research, namely the reporting of quantitative ion mobility measurements in the form of the gas-phase collision cross section (CCS), which has provided a common basis for comparison across different instrument platforms and offers a unique form of structural information, namely size and shape preferences of analytes in the absence of bulk solvent. This review surveys the over 24,000 CCS values reported from IM methods spanning the era between 1975 to 2015, which provides both a historical and analytical context for the contributions made thus far, as well as insight into the future directions that quantitative ion mobility measurements will have in the analytical sciences. The analysis was conducted in 2016, so CCS values reported in that year are purposely omitted. In another few years, a review of this scope will be intractable, as the number of CCS values which will be reported in the next three to five years is expected to exceed the total amount currently published in the literature.

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“…The term "run-on oligomer" (ROO) filament is used here to describe an assembly of an enzyme into a filament by the successive addition of enzymes at either end, and which, in principle, could extend indefinitely (8,16). ROO filament formation by SgrAI was first proposed in 2010 based on behavior in analytical ultracentrifugation and native gels (7) and subsequently using ion-mobility mass spectrometry (17). The enzymatic activity of SgrAI was found to be activated in the ROO and to possess an altered (expanded) DNA sequence specificity (7,8).…”

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The Need for Speed: Run-On Oligomer Filament Formation Provides Maximum Speed with Maximum Sequestration of Activity

Barahona

Basantes

Tompkins

et al. 2019

J Virol

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Here, we investigate an unusual antiviral mechanism developed in the bacterium Streptomyces griseus. SgrAI is a type II restriction endonuclease that forms run-on oligomer filaments when activated and possesses both accelerated DNA cleavage activity and expanded DNA sequence specificity. Mutations disrupting the run-on oligomer filament eliminate the robust antiphage activity of wild-type SgrAI, and the observation that even relatively modest disruptions completely abolish this anti-viral activity shows that the greater speed imparted by the run-on oligomer filament mechanism is critical to its biological function. Simulations of DNA cleavage by SgrAI uncover the origins of the kinetic advantage of this newly described mechanism of enzyme regulation over more conventional mechanisms, as well as the origin of the sequestering effect responsible for the protection of the host genome against damaging DNA cleavage activity of activated SgrAI. IMPORTANCE This work is motivated by an interest in understanding the characteristics and advantages of a relatively newly discovered enzyme mechanism involving filament formation. SgrAI is an enzyme responsible for protecting against viral infections in its host bacterium and was one of the first such enzymes shown to utilize such a mechanism. In this work, filament formation by SgrAI is disrupted, and the effects on the speed of the purified enzyme as well as its function in cells are measured. It was found that even small disruptions, which weaken but do not destroy filament formation, eliminate the ability of SgrAI to protect cells from viral infection, its normal biological function. Simulations of enzyme activity were also performed and show how filament formation can greatly speed up an enzyme’s activation compared to that of other known mechanisms, as well as to better localize its action to molecules of interest, such as invading phage DNA.

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Structural Analysis of Activated SgrAI–DNA Oligomers Using Ion Mobility Mass Spectrometry

Cited by 22 publications

References 66 publications

Allosteric Regulation of DNA Cleavage and Sequence-Specificity through Run-On Oligomerization

Allosteric Regulation of DNA Cleavage and Sequence-Specificity through Run-On Oligomerization

Ion Mobility Collision Cross Section Compendium

The Need for Speed: Run-On Oligomer Filament Formation Provides Maximum Speed with Maximum Sequestration of Activity

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