Markov State Models and Molecular Dynamics Simulations Provide Understanding of the Nucleotide-Dependent Dimerization-Based Activation of LRRK2 ROC Domain

Mutations in leucine-rich repeat kinase 2 (LRRK2) are recognized as the most frequent cause of Parkinson’s disease (PD). As a multidomain ROCO protein, LRRK2 is characterized by the presence of both a Ras-of-complex (ROC) GTPase domain and a kinase domain connected through the C-terminal of an ROC domain (COR). The bienzymatic ROC–COR–kinase catalytic triad indicated the potential role of GTPase domain in regulating kinase activity. However, as a functional GTPase, the detailed intrinsic regulation of the ROC activation cycle remains poorly understood. Here, combining extensive molecular dynamics simulations and Markov state models, we disclosed the dynamic structural rearrangement of ROC’s homodimer during nucleotide turnover. Our study revealed the coupling between dimerization extent and nucleotide-binding state, indicating a nucleotide-dependent dimerization-based activation scheme adopted by ROC GTPase. Furthermore, inspired by the well-known R1441C/G/H PD-relevant mutations within the ROC domain, we illuminated the potential allosteric molecular mechanism for its pathogenetic effects through enabling faster interconversion between inactive and active states, thus trapping ROC in a prolonged activated state, while the implicated allostery could provide further guidance for identification of regulatory allosteric pockets on the ROC complex. Our investigations illuminated the thermodynamics and kinetics of ROC homodimer during nucleotide-dependent activation for the first time and provided guidance for further exploiting ROC as therapeutic targets for controlling LRRK2 functionality in PD treatment.

CryoEM structure of the super-constricted two-start dynamin 1 filament

Dynamin belongs to the large GTPase superfamily, and mediates the fission of vesicles during endocytosis. Dynamin molecules are recruited to the neck of budding vesicles to assemble into a helical collar and to constrict the underlying membrane. Two helical forms were observed: the one-start helix in the constricted state and the two-start helix in the super-constricted state. Here we report the cryoEM structure of a super-constricted two-start dynamin 1 filament at 3.74 Å resolution. The two strands are joined by the conserved GTPase dimeric interface. In comparison with the one-start structure, a rotation around Hinge 1 is observed, essential for communicating the chemical power of the GTPase domain and the mechanical force of the Stalk and PH domain onto the underlying membrane. The Stalk interfaces are well conserved and serve as fulcrums for adapting to changing curvatures. Relative to one-start, small rotations per interface accumulate to bring a drastic change in the helical pitch. Elasticity theory rationalizes the diversity of dynamin helical symmetries and suggests corresponding functional significance.

Pathogenic DNM1 Gene Variant Presenting With Unusually Nonsevere Neurodevelopmental Phenotype: A Case Report

Background and ObjectivesTo date, all reports of pathogenic variants affecting the GTPase domain of the DNM1 gene have a clinically severe neurodevelopmental phenotype, including severe delays or intractable epilepsy. We describe a case with moderate developmental delays and self-resolved epilepsy.MethodsThe patient was followed by our neurology and genetics teams. After clinical examination and EEG to characterize the patient's presentation, we conducted etiologic workup including brain MRI, chromosomal microarray, genetic and metabolic investigations, and nerve conduction studies. Subsequently, we arranged an Intellectual Disability Plus Trio Panel.ResultsOur patient presented with seizures at 2 days old, requiring phenobarbital. She also had hypotonia, mild dysmorphic features, and mild ataxia. Although initial workup returned unremarkable, the trio gene panel identified a de novo heterozygous pathogenic missense variant in the DNM1 GTPase domain. Now 4 years old, she has been seizure-free for 3 years without ongoing treatment and has nonsevere developmental delays (e.g., ambulates independently and speaks 2-word phrases).DiscussionOur case confirms that not all individuals with DNM1 pathogenic variants, even affecting the GTPase domain, will present with intractable epilepsy or severe delays. Expanding the known clinical spectrum of dynamin-related neurodevelopmental disorder is crucial for patient prognostication and counseling.

DNM1L-Related Mitochondrial Fission Defects Presenting as Encephalopathy: A Case Report and Literature Review

Background: Mitochondrial dynamics, including mitochondrial fission and fusion, transport and distribution, biogenesis and degradation, are critical to neuronal function. The dynamin-1 like (DNM1L) gene encodes dynamin-related protein 1 (DRP1/DLP1), which is an evolutionarily conserved member of the dynamin family and is responsible for mitochondrial division. DNM1L variants can lead to mitochondrial fission dysfunction and neurological disorders.Methods: We report a case of DNM1L-related mitochondrial disease admitted to Tianjin Children's Hospital. We searched for similar reported cases in the PubMed database using the terms “DNM1L” and “mitochondrial,” reviewed recent literature to summarize the clinical and genetic characteristics, and analyzed genotype-phenotype correlations.Results: The patient presented with psychomotor retardation, motor disturbance (muscle weakness with paroxysmal hypermyotonia), and a de novo variant (c.116G>A, g.22229G>A, p.S39N) in the GTPase domain of DNM1L (reference sequence NM_012062), which has not previously been reported in the literature. This case was combined with an additional 35 cases identified in 20 relevant references in order to analyze a total of 36 patients. The male-to-female ratio was 1:1.06, and the median age of onset was 6 months (range, neonatal period to 9 years). The cardinal symptoms included psychomotor retardation in 77.8% (28/36), limb paralysis in 66.7% (18/27), dystonia in 82.8% (24/29), and epilepsy in 59.4% (19/32). The clinical manifestations of variants in the GTPase domain of DRP1 were milder than those identified in the middle domain.Conclusion: This case report describes a new variant of the DNM1L gene, and summarizes previously reported cases. Furthermore, the clinical phenotype and the genotype of DNM1L gene-associated mitochondrial disease was analyzed to improve the understanding of this disease.

Septin4 promotes cardiomyocytes apoptosis by enhancing the VHL-mediated degradation of HIF-1α

Septin4, a protein localized at mitochondrion, can promote cells apoptosis mainly by binding XIAP (X-linked inhibitors of apoptosis), however, nothing is known about the role and mechanism of Septin4 in cardiomyocytes apoptosis. Here in the current study, we report that HIF-1α (hypoxia-inducible factor 1 alpha) is a novel interacting protein with Septin4 at Septin4-GTPase domain. In addition, Septin4 enhances the binding between HIF-1α and the E3 ubiquitin ligase VHL (von Hippel-Lindau protein) to down-regulate HIF-1α, and by reducing cardio-protective factor HIF-1α levels, Septin4 aggravated the hypoxia-induced cardiomyocytes apoptosis. We believe these findings will be beneficial to provide effective strategies for clinical treatment of myocardial ischemia and the subsequent injury caused by myocardial hypoxia.

Domain dependent orchestrated regulation of bacterial growth, persistence and chemotaxis by an essential GTPase, CgtA, in Vibrio cholerae

SummaryCgtA, an evolutionarily conserved GTPase, associated with the 50S ribosome controls a broad spectrum of physiological processes in bacteria. It has three structural domains, viz., N-terminal domain (NTD), GTPase domain and C-terminal domain (CTD). CgtA regulates expression of several of genes during nutritional stress in Vibrio cholerae. The mechanism of transcriptional regulation by CgtA is unknown, though the NTD concomitantly with the GTPase domain participates in the process. Here, we show that the in vivo deletion of the 57 amino acids long CTD of CgtA GTPase of V. cholerae is dispensable for viability, contrary to the complete knockdown of cgtA gene. Slower growth was observed in cgtA knockdown strain with intermittent diauxic lags in minimal media than the CTD deleted strain. Irreversible defect in colony morphology was observed in the cells with CTD deletion. Resuscitation of persister cells occurred when nutritionally deprived complete cgtA knockdown cells after growing for longer periods were transferred to nutritionally enriched media. The motility of the cgtA knockdown strain was significantly reduced than the wild type cells. Furthermore, CTD deleted cells were also found to be defective in motility, but comparatively lower than cgtA knockdown cells. Elongated and slender V. cholerae cells were observed by SEM imaging upon cgtA depletion, whereas, upon CTD deletion cellular elongation did not occur. Based on our study here, we propose that the CTD of CgtA perceives the nutritional stress response, to which the NTD and GTPase responds.

A septin GTPase scaffold of dynein–dynactin motors triggers retrograde lysosome transport

The metabolic and signaling functions of lysosomes depend on their intracellular positioning and trafficking, but the underlying mechanisms are little understood. Here, we have discovered a novel septin GTPase–based mechanism for retrograde lysosome transport. We found that septin 9 (SEPT9) associates with lysosomes, promoting the perinuclear localization of lysosomes in a Rab7-independent manner. SEPT9 targeting to mitochondria and peroxisomes is sufficient to recruit dynein and cause perinuclear clustering. We show that SEPT9 interacts with both dynein and dynactin through its GTPase domain and N-terminal extension, respectively. Strikingly, SEPT9 associates preferentially with the dynein intermediate chain (DIC) in its GDP-bound state, which favors dimerization and assembly into septin multimers. In response to oxidative cell stress induced by arsenite, SEPT9 localization to lysosomes is enhanced, promoting the perinuclear clustering of lysosomes. We posit that septins function as GDP-activated scaffolds for the cooperative assembly of dynein–dynactin, providing an alternative mechanism of retrograde lysosome transport at steady state and during cellular adaptation to stress.

Crystal structure of the GDP-bound GTPase domain of Rab5a from Leishmania donovani

Eukaryotic Rab5s are highly conserved small GTPase-family proteins that are involved in the regulation of early endocytosis. Leishmania donovani Rab5a regulates the sorting of early endosomes that are involved in the uptake of essential nutrients through fluid-phase endocytosis. Here, the 1.80 Å resolution crystal structure of the N-terminal GTPase domain of L. donovani Rab5a in complex with GDP is presented. The crystal structure determination was enabled by the design of specific single-site mutations and two deletions that were made to stabilize the protein for previous NMR studies. The structure of LdRab5a shows the canonical GTPase fold, with a six-stranded central mixed β-sheet surrounded by five α-helices. The positions of the Switch I and Switch II loops confirm an open conformation, as expected in the absence of the γ-phosphate. However, in comparison to other GTP-bound and GDP-bound homologous proteins, the Switch I region traces a unique disposition in LdRab5a. One magnesium ion is bound to the protein at the GTP-binding site. Molecular-dynamics simulations indicate that the GDP-bound structure exhibits higher stability than the apo structure. The GDP-bound LdRab5a structure presented here will aid in efforts to unravel its interactions with its regulators, including the guanine nucleotide-exchange factor, and will lay the foundation for a structure-based search for specific inhibitors

Rab34 GTPase mediates ciliary membrane biogenesis in the intracellular ciliogenesis pathway

Primary cilia form by two pathways: an extracellular pathway in which the cilium grows out from the cell surface and an intracellular pathway in which the nascent cilium forms inside the cell. Here we identify the GTPase Rab34 as a selective mediator of intracellular ciliogenesis. We find that Rab34 is required for formation of the ciliary vesicle at the mother centriole and that Rab34 marks the ciliary sheath, a unique sub-domain of assembling intracellular cilia. Rab34 activity is modulated by divergent residues within its GTPase domain, and ciliogenesis requires GTP binding and turnover by Rab34. Because Rab34 is found on assembly intermediates that are unique to intracellular ciliogenesis, we tested its role in the extracellular pathway used by MDCK cells. Consistent with Rab34 acting specifically in the intracellular pathway, MDCK cells ciliate independently of Rab34 and paralog Rab36. Together, these findings reveal a new context-specific molecular requirement for ciliary membrane biogenesis.