scholarly journals Auto-regulation of Rab5 GEF activity in Rabex5 by allosteric structural changes, catalytic core dynamics and ubiquitin binding

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Janelle Lauer ◽  
Sandra Segeletz ◽  
Alice Cezanne ◽  
Giambattista Guaitoli ◽  
Francesco Raimondi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Structural Changes ◽  
Structural Model ◽  
Regulation Mechanism ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Catalytic Core ◽  
Exchange Mass Spectrometry ◽  
C Terminus ◽  
Ubiquitin Binding

Intracellular trafficking depends on the function of Rab GTPases, whose activation is regulated by guanine exchange factors (GEFs). The Rab5 GEF, Rabex5, was previously proposed to be auto-inhibited by its C-terminus. Here, we studied full-length Rabex5 and Rabaptin5 proteins as well as domain deletion Rabex5 mutants using hydrogen deuterium exchange mass spectrometry. We generated a structural model of Rabex5, using chemical cross-linking mass spectrometry and integrative modeling techniques. By correlating structural changes with nucleotide exchange activity for each construct, we uncovered new auto-regulatory roles for the ubiquitin binding domains and the Linker connecting those domains to the catalytic core of Rabex5. We further provide evidence that enhanced dynamics in the catalytic core are linked to catalysis. Our results suggest a more complex auto-regulation mechanism than previously thought and imply that ubiquitin binding serves not only to position Rabex5 but to also control its Rab5 GEF activity through allosteric structural alterations.

scholarly journals Allosteric Structural Alterations and Auto-regulation of Rab5 GEF Activity in Rabex5

2019 ◽  
Author(s):  
Janelle Lauer ◽  
Alice Cezanne ◽  
Giambattista Guaitoli ◽  
Francesco Raimondi ◽  
Marc Gentzel ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Structural Changes ◽  
Structural Model ◽  
Regulation Mechanism ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Catalytic Core ◽  
Structural Alterations ◽  
Exchange Mass Spectrometry ◽  
Ubiquitin Binding

AbstractIntracellular trafficking depends on the function of Rab GTPases, whose activation is regulated by guanine exchange factors (GEFs). The Rab5 GEF, Rabex5, was previously proposed to be autoinhibited by its C-terminus. Here, we studied full-length Rabex5 and Rabaptin5 proteins as well as domain deletion Rabex5 mutants using hydrogen deuterium exchange mass spectrometry. We generated a structural model of Rabex5, using chemical crosslinking mass spectrometry and integrative modeling techniques. Our results are inconsistent with the previous model of autoinhibition. By correlating structural changes with nucleotide exchange activity for each construct, we uncovered new auto-regulatory roles for the Ubiquitin binding domains and the Linker connecting those domains to the catalytic core of Rabex5. Our results suggest a more complex auto-regulation mechanism than previously thought and imply that Ubiquitin binding serves not only to position Rabex5 but to also control its Rab5 GEF activity through allosteric structural alterations.

scholarly journals Biochemical insight into novel Rab-GEF activity of the mammalian TRAPPIII complex

2021 ◽  
Author(s):  
Noah J Harris ◽  
Meredith L. Jenkins ◽  
Udit Dalwadi ◽  
Kaelin D Fleming ◽  
Sung-Eun Nam ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Membrane Trafficking ◽  
Lipid Membranes ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Exchange Mass Spectrometry ◽  
Accessory Subunits ◽  
Hdx Ms ◽  
Insight Into

Transport Protein Particle complexes (TRAPP) are evolutionarily conserved regulators of membrane trafficking, with this mediated by their guanine nucleotide exchange factor (GEF) activity towards Rab GTPases. In metazoans evidence suggests that two different TRAPP complexes exist, TRAPPII and TRAPPIII. These two complexes share a common core of subunits, with complex specific subunits (TRAPPC9 and TRAPPC10 in TRAPPII and TRAPPC8, TRAPPC11, TRAPPC12, TRAPPC13 in TRAPPIII). TRAPPII and TRAPPIII have distinct specificity for GEF activity towards Rabs, with TRAPPIII acting on Rab1, and TRAPPII acting on Rab1 and Rab11. The molecular basis for how these complex specific subunits alter GEF activity towards Rab GTPases is unknown. Here we have used a combination of biochemical assays, hydrogen deuterium exchange mass spectrometry (HDX-MS) and electron microscopy to examine the regulation of TRAPPII and TRAPPIIII complexes in solution and on membranes. GEF assays revealed that the TRAPPIII has GEF activity against Rab1 and Rab43, with no detectable activity against the other 18 Rabs tested. The TRAPPIII complex had significant differences in protein dynamics at the Rab binding site compared to TRAPPII, potentially indicating an important role of accessory subunits in altering the active site of TRAPP complexes. Both the TRAPPII and TRAPPIII complexes had enhanced GEF activity on lipid membranes, with HDX-MS revealing numerous conformational changes that accompany membrane association. HDX-MS also identified a membrane binding site in TRAPPC8. Collectively, our results provide insight into the functions of TRAPP complexes and how they can achieve Rab specificity.

scholarly journals Mono- and Biallelic Protein-Truncating Variants in Alpha-Actinin 2 Cause Cardiomyopathy Through Distinct Mechanisms

2021 ◽  
Author(s):  
Malene E. Lindholm ◽  
David Jimenez-Morales ◽  
Han Zhu ◽  
Kinya Seo ◽  
David Amar ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Structural Changes ◽  
Affinity Purification ◽  
Protein Protein Interaction ◽  
Cellular Hypertrophy ◽  
C Terminus ◽  
Associated Proteins ◽  
Cardiac Sarcomeres ◽  
Affinity Purification Mass Spectrometry

Background: ACTN2 (alpha-actinin 2) anchors actin within cardiac sarcomeres. The mechanisms linking ACTN2 mutations to myocardial disease phenotypes are unknown. Here, we characterize patients with novel ACTN2 mutations to reveal insights into the physiological function of ACTN2. Methods: Patients harboring ACTN2 protein-truncating variants were identified using a custom mutation pipeline. In patient-derived iPSC-cardiomyocytes, we investigated transcriptional profiles using RNA sequencing, contractile properties using video-based edge detection, and cellular hypertrophy using immunohistochemistry. Structural changes were analyzed through electron microscopy. For mechanistic studies, we used coimmunoprecipitation for ACTN2, followed by mass-spectrometry to investigate protein-protein interaction, and protein tagging followed by confocal microscopy to investigate introduction of truncated ACTN2 into the sarcomeres. Results: Patient-derived iPSC-cardiomyocytes were hypertrophic, displayed sarcomeric structural disarray, impaired contractility, and aberrant Ca 2+ -signaling. In heterozygous indel cells, the truncated protein incorporates into cardiac sarcomeres, leading to aberrant Z-disc ultrastructure. In homozygous stop-gain cells, affinity-purification mass-spectrometry reveals an intricate ACTN2 interactome with sarcomere and sarcolemma-associated proteins. Loss of the C-terminus of ACTN2 disrupts interaction with ACTN1 and GJA1, 2 sarcolemma-associated proteins, which may contribute to the clinical arrhythmic and relaxation defects. The causality of the stop-gain mutation was verified using CRISPR-Cas9 gene editing. Conclusions: Together, these data advance our understanding of the role of ACTN2 in the human heart and establish recessive inheritance of ACTN2 truncation as causative of disease.

scholarly journals Structural dynamics of the human COP9 signalosome revealed by cross-linking mass spectrometry and integrative modeling

2020 ◽  
Vol 117 (8) ◽  
pp. 4088-4098 ◽  
Author(s):  
Craig Gutierrez ◽  
Ilan E. Chemmama ◽  
Haibin Mao ◽  
Clinton Yu ◽  
Ignacia Echeverria ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Structural Dynamics ◽  
Structural Changes ◽  
Structural Model ◽  
Protein Complexes ◽  
Cop9 Signalosome ◽  
Cross Linking ◽  
E3 Ligases ◽  
Protein Ubiquitination ◽  
Mass Spectometry

The COP9 signalosome (CSN) is an evolutionarily conserved eight-subunit (CSN1–8) protein complex that controls protein ubiquitination by deneddylating Cullin-RING E3 ligases (CRLs). The activation and function of CSN hinges on its structural dynamics, which has been challenging to decipher by conventional tools. Here, we have developed a multichemistry cross-linking mass spectrometry approach enabled by three mass spectometry-cleavable cross-linkers to generate highly reliable cross-link data. We applied this approach with integrative structure modeling to determine the interaction and structural dynamics of CSN with the recently discovered ninth subunit, CSN9, in solution. Our results determined the localization of CSN9 binding sites and revealed CSN9-dependent structural changes of CSN. Together with biochemical analysis, we propose a structural model in which CSN9 binding triggers CSN to adopt a configuration that facilitates CSN–CRL interactions, thereby augmenting CSN deneddylase activity. Our integrative structure analysis workflow can be generalized to define in-solution architectures of dynamic protein complexes that remain inaccessible to other approaches.

scholarly journals Dynamic structural changes during complement C3 activation analyzed by hydrogen/deuterium exchange mass spectrometry

2008 ◽  
Vol 45 (11) ◽  
pp. 3142-3151 ◽  
Author(s):  
Michael C. Schuster ◽  
Daniel Ricklin ◽  
Krisztián Papp ◽  
Kathleen S. Molnar ◽  
Stephen J. Coales ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Structural Changes ◽  
Deuterium Exchange ◽  
Complement C3 ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry

scholarly journals Author response: Auto-regulation of Rab5 GEF activity in Rabex5 by allosteric structural changes, catalytic core dynamics and ubiquitin binding

2019 ◽  
Author(s):  
Janelle Lauer ◽  
Sandra Segeletz ◽  
Alice Cezanne ◽  
Giambattista Guaitoli ◽  
Francesco Raimondi ◽  
...  
Keyword(s):  
Structural Changes ◽  
Author Response ◽  
Catalytic Core ◽  
Core Dynamics ◽  
Ubiquitin Binding

scholarly journals Structural Characterization of Full-Length Human Dehydrodolichyl Diphosphate Synthase Using an Integrative Computational and Experimental Approach

Biomolecules ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 660 ◽  
Author(s):  
Michal Lisnyansky Bar-El ◽  
Su Youn Lee ◽  
Ah Young Ki ◽  
Noa Kapelushnik ◽  
Anat Loewenstein ◽  
...  
Keyword(s):  
Structural Model ◽  
Catalytic Domain ◽  
Clinical Importance ◽  
Protein Glycosylation ◽  
Full Length ◽  
Size Exclusion ◽  
Scattering Data ◽  
Exchange Mass Spectrometry ◽  
Functional Studies ◽  
C Terminus

Dehydrodolichyl diphosphate synthase (DHDDS) is the catalytic subunit of the heteromeric human cis-prenyltransferase complex, synthesizing the glycosyl carrier precursor for N-linked protein glycosylation. Consistent with the important role of N-glycosylation in protein biogenesis, DHDDS mutations result in human diseases. Importantly, DHDDS encompasses a C-terminal region, which does not converge with any known conserved domains. Therefore, despite the clinical importance of DHDDS, our understating of its structure–function relations remains poor. Here, we provide a structural model for the full-length human DHDDS using a multidisciplinary experimental and computational approach. Size-exclusion chromatography multi-angle light scattering revealed that DHDDS forms a monodisperse homodimer in solution. Enzyme kinetics assays revealed that it exhibits catalytic activity, although reduced compared to that reported for the intact heteromeric complex. Our model suggests that the DHDDS C-terminus forms a helix–turn–helix motif, tightly packed against the core catalytic domain. This model is consistent with small-angle X-ray scattering data, indicating that the full-length DHDDS maintains a similar conformation in solution. Moreover, hydrogen–deuterium exchange mass-spectrometry experiments show time-dependent deuterium uptake in the C-terminal domain, consistent with its overall folded state. Finally, we provide a model for the DHDDS–NgBR heterodimer, offering a structural framework for future structural and functional studies of the complex.

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