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Article ; Online: The MIDAS domain of AAA mechanoenzyme Mdn1 forms catch bonds with two different substrates.

Mickolajczyk, Keith J / Olinares, Paul Dominic B / Chait, Brian T / Liu, Shixin / Kapoor, Tarun M

2022 Volume 11

Abstract: Catch bonds are a form of mechanoregulation wherein protein-ligand interactions are strengthened by the application of dissociative tension. Currently, the best-characterized examples of catch bonds are between single protein-ligand pairs. The essential ... ...

Abstract	Catch bonds are a form of mechanoregulation wherein protein-ligand interactions are strengthened by the application of dissociative tension. Currently, the best-characterized examples of catch bonds are between single protein-ligand pairs. The essential AAA (ATPase associated with diverse cellular activities) mechanoenzyme Mdn1 drives at least two separate steps in ribosome biogenesis, using its MIDAS domain to extract the ubiquitin-like (UBL) domain-containing proteins Rsa4 and Ytm1 from ribosomal precursors. However, it must subsequently release these assembly factors to reinitiate the enzymatic cycle. The mechanism underlying the switching of the MIDAS-UBL interaction between strongly and weakly bound states is unknown. Here, we use optical tweezers to investigate the force dependence of MIDAS-UBL binding. Parallel experiments with Rsa4 and Ytm1 show that forces up to ~4 pN, matching the magnitude of force produced by AAA proteins similar to Mdn1, enhance the MIDAS domain binding lifetime up to 10-fold, and higher forces accelerate dissociation. Together, our studies indicate that Mdn1's MIDAS domain can form catch bonds with more than one UBL substrate, and provide insights into how mechanoregulation may contribute to the Mdn1 enzymatic cycle during ribosome biogenesis.
MeSH term(s)	ATPases Associated with Diverse Cellular Activities/metabolism ; Binding Sites ; Ligands ; Optical Tweezers ; Organelle Biogenesis ; Protein Binding ; Protein Domains ; Ribosome Subunits, Large, Eukaryotic/metabolism ; Ribosomes/physiology ; Saccharomyces cerevisiae Proteins ; Schizosaccharomyces pombe Proteins/metabolism ; Single Molecule Imaging ; Ubiquitin/genetics
Chemical Substances	Ligands ; Saccharomyces cerevisiae Proteins ; Schizosaccharomyces pombe Proteins ; Ubiquitin ; MDN1 protein, S cerevisiae (EC 3.6.1.3) ; Mdn1 protein, S pombe (EC 3.6.1.3) ; ATPases Associated with Diverse Cellular Activities (EC 3.6.4.-)
Language	English
Publishing date	2022-02-11
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2687154-3
ISSN	2050-084X ; 2050-084X
ISSN (online)	2050-084X
ISSN	2050-084X
DOI	10.7554/eLife.73534
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Article: Differential dynamics specify MeCP2 function at methylated DNA and nucleosomes.

Chua, Gabriella N L / Watters, John W / Olinares, Paul Dominic B / Luo, Joshua A / Chait, Brian T / Liu, Shixin

bioRxiv : the preprint server for biology

2023

Abstract: Methyl-CpG-binding protein 2 (MeCP2) is an essential chromatin-binding protein whose mutations cause Rett syndrome (RTT), a leading cause of monogenic intellectual disabilities in females. Despite its significant biomedical relevance, the mechanism by ... ...

Abstract	Methyl-CpG-binding protein 2 (MeCP2) is an essential chromatin-binding protein whose mutations cause Rett syndrome (RTT), a leading cause of monogenic intellectual disabilities in females. Despite its significant biomedical relevance, the mechanism by which MeCP2 navigates the chromatin epigenetic landscape to regulate chromatin structure and gene expression remains unclear. Here, we used correlative single-molecule fluorescence and force microscopy to directly visualize the distribution and dynamics of MeCP2 on a variety of DNA and chromatin substrates. We found that MeCP2 exhibits differential diffusion dynamics when bound to unmethylated and methylated bare DNA. Moreover, we discovered that MeCP2 preferentially binds nucleosomes within the context of chromatinized DNA and stabilizes them from mechanical perturbation. The distinct behaviors of MeCP2 at bare DNA and nucleosomes also specify its ability to recruit TBLR1, a core component of the NCoR1/2 co-repressor complex. We further examined several RTT mutations and found that they disrupt different aspects of the MeCP2-chromatin interaction, rationalizing the heterogeneous nature of the disease. Our work reveals the biophysical basis for MeCP2's methylation-dependent activities and suggests a nucleosome-centric model for its genomic distribution and gene repressive functions. These insights provide a framework for delineating the multifaceted functions of MeCP2 and aid in our understanding of the molecular mechanisms of RTT.
Language	English
Publishing date	2023-06-05
Publishing country	United States
Document type	Preprint
DOI	10.1101/2023.06.02.543478
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Article ; Online: A nucleotide binding-independent role for γ-tubulin in microtubule capping and cell division.

Berman, Adi Y / Wieczorek, Michal / Aher, Amol / Olinares, Paul Dominic B / Chait, Brian T / Kapoor, Tarun M

The Journal of cell biology

2023 Volume 222, Issue 3

Abstract: The γ-tubulin ring complex (γ-TuRC) has essential roles in centrosomal and non-centrosomal microtubule organization during vertebrate mitosis. While there have been important advances in understanding γ-TuRC-dependent microtubule nucleation, γ-TuRC ... ...

Abstract	The γ-tubulin ring complex (γ-TuRC) has essential roles in centrosomal and non-centrosomal microtubule organization during vertebrate mitosis. While there have been important advances in understanding γ-TuRC-dependent microtubule nucleation, γ-TuRC capping of microtubule minus-ends remains poorly characterized. Here, we utilized biochemical reconstitutions and cellular assays to characterize the human γ-TuRC's capping activity. Single filament assays showed that the γ-TuRC remained associated with a nucleated microtubule for tens of minutes. In contrast, caps at dynamic microtubule minus-ends displayed lifetimes of ∼1 min. Reconstituted γ-TuRCs with nucleotide-binding deficient γ-tubulin (γ-tubulinΔGTP) formed ring-shaped complexes that did not nucleate microtubules but capped microtubule minus-ends with lifetimes similar to those measured for wild-type complexes. In dividing cells, microtubule regrowth assays revealed that while knockdown of γ-tubulin suppressed non-centrosomal microtubule formation, add-back of γ-tubulinΔGTP could substantially restore this process. Our results suggest that γ-TuRC capping is a nucleotide-binding-independent activity that plays a role in non-centrosomal microtubule organization during cell division.
MeSH term(s)	Humans ; Tubulin/chemistry ; Microtubule-Associated Proteins/genetics ; Microtubules/chemistry ; Microtubule-Organizing Center ; Cell Division
Chemical Substances	Tubulin ; Microtubule-Associated Proteins
Language	English
Publishing date	2023-01-25
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	218154-x
ISSN	1540-8140 ; 0021-9525
ISSN (online)	1540-8140
ISSN	0021-9525
DOI	10.1083/jcb.202204102
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Article ; Online: Native Mass Spectrometry Analysis of Affinity-Captured Endogenous Yeast RNA Exosome Complexes.

Olinares, Paul Dominic B / Chait, Brian T

Methods in molecular biology (Clifton, N.J.)

2019 Volume 2062, Page(s) 357–382

Abstract: Native mass spectrometry (MS) enables direct mass measurement of intact protein assemblies generating relevant subunit composition and stoichiometry information. Combined with cross-linking and structural data, native MS-derived information is crucial ... ...

Abstract	Native mass spectrometry (MS) enables direct mass measurement of intact protein assemblies generating relevant subunit composition and stoichiometry information. Combined with cross-linking and structural data, native MS-derived information is crucial for elucidating the architecture of macromolecular assemblies by integrative structural methods. The exosome complex from budding yeast was among the first endogenous protein complexes to be affinity isolated and subsequently characterized by this technique, providing improved understanding of its composition and structure. We present a protocol that couples efficient affinity capture of yeast exosome complexes and sensitive native MS analysis, including rapid affinity isolation of the endogenous exosome complex from cryolysed yeast cells, elution in nondenaturing conditions by protease cleavage, depletion of the protease, buffer exchange, and native MS measurements using an Orbitrap-based instrument (Exactive Plus EMR).
MeSH term(s)	Chromatography, Affinity/methods ; Exosome Multienzyme Ribonuclease Complex/metabolism ; Macromolecular Substances/metabolism ; Saccharomyces cerevisiae/metabolism ; Saccharomyces cerevisiae Proteins/metabolism ; Tandem Mass Spectrometry/methods
Chemical Substances	Macromolecular Substances ; Saccharomyces cerevisiae Proteins ; Exosome Multienzyme Ribonuclease Complex (EC 3.1.-)
Language	English
Publishing date	2019-11-19
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-4939-9822-7_17
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Article ; Online: Structural basis of transcriptional regulation by a nascent RNA element, HK022 putRNA.

Hwang, Seungha / Olinares, Paul Dominic B / Lee, Jimin / Kim, Jinwoo / Chait, Brian T / King, Rodney A / Kang, Jin Young

Nature communications

2022 Volume 13, Issue 1, Page(s) 4668

Abstract: Transcription, in which RNA polymerases (RNAPs) produce RNA from DNA, is the first step of gene expression. As such, it is highly regulated either by trans-elements like protein factors and/or by cis-elements like specific sequences on the DNA. Lambdoid ... ...

Abstract	Transcription, in which RNA polymerases (RNAPs) produce RNA from DNA, is the first step of gene expression. As such, it is highly regulated either by trans-elements like protein factors and/or by cis-elements like specific sequences on the DNA. Lambdoid phage HK022 contains a cis-element, put, which suppresses pausing and termination during transcription of the early phage genes. The putRNA transcript solely performs the anti-pausing/termination activities by interacting directly with the E.coli RNAP elongation complex (EC) by an unknown structural mechanism. In this study, we reconstituted putRNA-associated ECs and determined the structures using cryo-electron microscopy. The determined structures of putRNA-associated EC, putRNA-absent EC, and σ
MeSH term(s)	Bacteriophage lambda/genetics ; Cryoelectron Microscopy ; DNA/metabolism ; DNA-Directed RNA Polymerases/metabolism ; Escherichia coli/genetics ; Escherichia coli/virology ; RNA/genetics ; RNA/metabolism ; Transcription, Genetic
Chemical Substances	RNA (63231-63-0) ; DNA (9007-49-2) ; DNA-Directed RNA Polymerases (EC 2.7.7.6)
Language	English
Publishing date	2022-08-15
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-022-32315-y
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Article: Structural and functional insights into the enzymatic plasticity of the SARS-CoV-2 NiRAN Domain.

Small, Gabriel I / Fedorova, Olga / Olinares, Paul Dominic B / Chandanani, Joshua / Banerjee, Anoosha / Choi, Young Joo / Molina, Henrik / Chait, Brian / Darst, Seth A / Campbell, Elizabeth A

bioRxiv : the preprint server for biology

2023

Abstract: The enzymatic activity of the SARS-CoV-2 nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain is essential for viral propagation, with three distinct activities associated with modification of the nsp9 N-terminus, NMPylation, RNAylation, and ... ...

Abstract	The enzymatic activity of the SARS-CoV-2 nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain is essential for viral propagation, with three distinct activities associated with modification of the nsp9 N-terminus, NMPylation, RNAylation, and deRNAylation/capping via a GDP-polyribonucleotidyltransferase reaction. The latter two activities comprise an unconventional mechanism for initiating viral RNA 5'-cap formation, while the role of NMPylation is unclear. The structural mechanisms for these diverse enzymatic activities have not been properly delineated. Here we determine high-resolution cryo-electron microscopy structures of catalytic intermediates for the NMPylation and deRNAylation/capping reactions, revealing diverse nucleotide binding poses and divalent metal ion coordination sites to promote its repertoire of activities. The deRNAylation/capping structure explains why GDP is a preferred substrate for the capping reaction over GTP. Altogether, these findings enhance our understanding of the promiscuous coronaviral NiRAN domain, a therapeutic target, and provide an accurate structural platform for drug development.
Language	English
Publishing date	2023-09-26
Publishing country	United States
Document type	Preprint
DOI	10.1101/2023.09.25.558837
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Article ; Online: Shelterin is a dimeric complex with extensive structural heterogeneity.

Zinder, John C / Olinares, Paul Dominic B / Svetlov, Vladimir / Bush, Martin W / Nudler, Evgeny / Chait, Brian T / Walz, Thomas / de Lange, Titia

Proceedings of the National Academy of Sciences of the United States of America

2022 Volume 119, Issue 31, Page(s) e2201662119

Abstract: Human shelterin is a six-subunit complex-composed of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1-that binds telomeres, protects them from the DNA-damage response, and regulates the maintenance of telomeric DNA. Although high-resolution structures have been ... ...

Abstract	Human shelterin is a six-subunit complex-composed of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1-that binds telomeres, protects them from the DNA-damage response, and regulates the maintenance of telomeric DNA. Although high-resolution structures have been generated of the individual structured domains within shelterin, the architecture and stoichiometry of the full complex are currently unknown. Here, we report the purification of shelterin subcomplexes and reconstitution of the entire complex using full-length, recombinant subunits. By combining negative-stain electron microscopy (EM), cross-linking mass spectrometry (XLMS), AlphaFold modeling, mass photometry, and native mass spectrometry (MS), we obtain stoichiometries as well as domain-scale architectures of shelterin subcomplexes and determine that they feature extensive conformational heterogeneity. For POT1/TPP1 and POT1/TPP1/TIN2, we observe high variability in the positioning of the POT1 DNA-binding domain, the TPP1 oligonucleotide/oligosaccharide-binding (OB) fold, and the TIN2 TRFH domain with respect to the C-terminal domains of POT1. Truncation of unstructured linker regions in TIN2, TPP1, and POT1 did not reduce the conformational variability of the heterotrimer. Shelterin and TRF1-containing subcomplexes form fully dimeric stoichiometries, even in the absence of DNA substrates. Shelterin and its subcomplexes showed extensive conformational variability, regardless of the presence of DNA substrates. We conclude that shelterin adopts a multitude of conformations and argue that its unusual architectural variability is beneficial for its many functions at telomeres.
MeSH term(s)	Humans ; Mass Spectrometry ; Microscopy, Electron ; Protein Domains ; Shelterin Complex/chemistry
Chemical Substances	Shelterin Complex
Language	English
Publishing date	2022-07-26
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.2201662119
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Zs.A 1148: Show issues

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Article ; Online: Structural and functional insights into the enzymatic plasticity of the SARS-CoV-2 NiRAN domain.

Small, Gabriel I / Fedorova, Olga / Olinares, Paul Dominic B / Chandanani, Joshua / Banerjee, Anoosha / Choi, Young Joo / Molina, Henrik / Chait, Brian T / Darst, Seth A / Campbell, Elizabeth A

Molecular cell

2023 Volume 83, Issue 21, Page(s) 3921–3930.e7

Abstract	The enzymatic activity of the SARS-CoV-2 nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain is essential for viral propagation, with three distinct activities associated with modification of the nsp9 N terminus, NMPylation, RNAylation, and deRNAylation/capping via a GDP-polyribonucleotidyltransferase reaction. The latter two activities comprise an unconventional mechanism for initiating viral RNA 5' cap formation, while the role of NMPylation is unclear. The structural mechanisms for these diverse enzymatic activities have not been properly delineated. Here, we determine high-resolution cryoelectron microscopy (cryo-EM) structures of catalytic intermediates for the NMPylation and deRNAylation/capping reactions, revealing diverse nucleotide binding poses and divalent metal ion coordination sites to promote its repertoire of activities. The deRNAylation/capping structure explains why GDP is a preferred substrate for the capping reaction over GTP. Altogether, these findings enhance our understanding of the promiscuous coronaviral NiRAN domain, a therapeutic target, and provide an accurate structural platform for drug development.
MeSH term(s)	Humans ; Nucleotidyltransferases/metabolism ; SARS-CoV-2/genetics ; SARS-CoV-2/metabolism ; Cryoelectron Microscopy ; COVID-19 ; RNA, Viral/genetics
Chemical Substances	Nucleotidyltransferases (EC 2.7.7.-) ; RNA, Viral
Language	English
Publishing date	2023-10-26
Publishing country	United States
Document type	Journal Article
ZDB-ID	1415236-8
ISSN	1097-4164 ; 1097-2765
ISSN (online)	1097-4164
ISSN	1097-2765
DOI	10.1016/j.molcel.2023.10.001
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Zs.A 5055: Show issues

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Article ; Online: Structural basis of substrate recognition by a polypeptide processing and secretion transporter.

Kieuvongngam, Virapat / Olinares, Paul Dominic B / Palillo, Anthony / Oldham, Michael L / Chait, Brian T / Chen, Jue

eLife

2020 Volume 9

Abstract: The peptidase-containing ATP-binding cassette transporters (PCATs) are unique members of the ABC transporter family that proteolytically process and export peptides and proteins. Each PCAT contains two peptidase domains that cleave off the secretion ... ...

Abstract	The peptidase-containing ATP-binding cassette transporters (PCATs) are unique members of the ABC transporter family that proteolytically process and export peptides and proteins. Each PCAT contains two peptidase domains that cleave off the secretion signal, two transmembrane domains forming a translocation pathway, and two nucleotide-binding domains that hydrolyze ATP. Previously the crystal structures of a PCAT from
MeSH term(s)	ATP-Binding Cassette Transporters/chemistry ; Adenosine Triphosphate/metabolism ; Bacterial Proteins/chemistry ; Biological Transport ; Clostridium thermocellum/chemistry ; Hydrolysis ; Protein Sorting Signals
Chemical Substances	ATP-Binding Cassette Transporters ; Bacterial Proteins ; Protein Sorting Signals ; Adenosine Triphosphate (8L70Q75FXE)
Language	English
Publishing date	2020-01-14
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2687154-3
ISSN	2050-084X ; 2050-084X
ISSN (online)	2050-084X
ISSN	2050-084X
DOI	10.7554/eLife.51492
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Article ; Online: The MIDAS domain of AAA mechanoenzyme Mdn1 forms catch bonds with two different substrates

Keith J Mickolajczyk / Paul Dominic B Olinares / Brian T Chait / Shixin Liu / Tarun M Kapoor

eLife, Vol

2022 Volume 11

Abstract	Catch bonds are a form of mechanoregulation wherein protein-ligand interactions are strengthened by the application of dissociative tension. Currently, the best-characterized examples of catch bonds are between single protein-ligand pairs. The essential AAA (ATPase associated with diverse cellular activities) mechanoenzyme Mdn1 drives at least two separate steps in ribosome biogenesis, using its MIDAS domain to extract the ubiquitin-like (UBL) domain-containing proteins Rsa4 and Ytm1 from ribosomal precursors. However, it must subsequently release these assembly factors to reinitiate the enzymatic cycle. The mechanism underlying the switching of the MIDAS-UBL interaction between strongly and weakly bound states is unknown. Here, we use optical tweezers to investigate the force dependence of MIDAS-UBL binding. Parallel experiments with Rsa4 and Ytm1 show that forces up to ~4 pN, matching the magnitude of force produced by AAA proteins similar to Mdn1, enhance the MIDAS domain binding lifetime up to 10-fold, and higher forces accelerate dissociation. Together, our studies indicate that Mdn1’s MIDAS domain can form catch bonds with more than one UBL substrate, and provide insights into how mechanoregulation may contribute to the Mdn1 enzymatic cycle during ribosome biogenesis.
Keywords	catch bond ; optical tweezers ; ribosome biogenesis ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
Subject code	500
Language	English
Publishing date	2022-02-01T00:00:00Z
Publisher	eLife Sciences Publications Ltd
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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