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Sundaramoorthy, R.

Publications and source records attributed to Sundaramoorthy, R..

11 recordsLinked to original sources

Dissecting the interactions of the ISG15-USP18-STAT2 inhibitory complex

The suppression of type I interferon (IFN) signalling by the ISG15-USP18-STAT2 inhibitory complex (ISG15 IC) is an established regulatory mechanism of the antiviral response. However, a molecular understanding of how the ISG15 IC forms to suppress IFN signalling is still emerging. Here, we use AlphaFold modelling in conjunction with biochemical and biophysical approaches to elucidate the interactions of this multiprotein assembly. Our analysis identified a unique STAT2 binding loop (SBL) in USP18, which is critical for the USP18-STAT2 association. Further biochemical characterisation through site-directed mutagenesis confirmed the importance of residues within and surrounding the SBL, enabling the design of mutants with both increased and decreased binding affinities. Moreover, several USP18 and STAT2 patient mutations severely disrupted this interaction. Lastly, using influenza B virus (IBV) and Zika virus (ZIKV) proteins, we investigated the influence of these viral effector proteins on these interactions. Taken together, these results provide much-needed insights into a key aspect of IFN signalling control.

biochemistry↗

Dimerization and Threonine-Dependent Stabilization Govern Human YRDC Catalysis

Addition of N6-threonylcarbamoyladenosine occurs at position 37 (tA37) of five mitochondrial tRNA species, influencing translation fidelity and efficiency. Mutations in YRDC, the enzyme catalyzing the first step in tA37 synthesis, cause severe neurological and renal diseases in humans, including in Galloway-Mowatt disease (GAMOS). YRDC generates threonylcarbamoyl-AMP (TC-AMP) using ATP, threonine and bicarbonate as substrates, yet their binding order, residues involved in amino-acid selectivity, and precisely how mutations contribute to disease remain unclear. Here, we combine protein biophysics, mass spectrometry, NMR, mutagenesis and kinetics to define how the human enzyme operates. Differential scanning fluorimetry and circular dichroism identify L-threonine as the gatekeeper ligand. It binds with millimolar affinity, stabilizing YRDC and enhancing ATP binding nearly fortyfold, enabling bicarbonate association. Several amino acids can form aminoacylcarbamoyl-AMP adducts in vitro, but threonine yields superior protein stabilization and product formation. Native mass spectrometry and single-molecule mass photometry corroborate YRDCs dimeric state. GAMOS-linked mutations destabilize folding, disrupting dimerization and uncoupling substrate binding from catalysis, explaining functional losses. Supported by kinetic data and NMR, we propose a chemical mechanism for the reaction and identify dimerization and conformational stability as control points for activity. This work provides a foundation for designing selective YRDC inhibitors or enhancers.

biochemistry↗

Broad substrate scope C-C oxidation in cyclodipeptides catalysed by a flavin-dependent filament

Cyclic dipeptides are produced by organisms in all domains of life. Many possess biological activities as anticancer and antimicrobial compounds. Oxidations are frequently present in these biologically active peptides. C-C bond oxidation is catalysed by tailoring enzymes, including cyclodipeptide oxidases. These flavin-dependent enzymes are underexplored due to their complex three-dimensional arrangement involving multiple copies of two distinct small subunits and unclear mechanistic features underlying substrate selection and catalysis. Here, we determined the structure and mechanism of the cyclodipeptide oxidase from the halophile Nocardiopsis dassonvillei (NdasCDO), part of the biosynthetic pathway for nocazine natural products. We show NdasCDO forms filaments in solution with a covalently bound FMN cofactor in the interface between three distinct subunits. The enzyme is promiscuous, using many cyclic dipeptides as substrates in a distributive manner. The reaction is optimal at high pH, involving the formation of a radical intermediate. Pre-steady state kinetics, a sizeable solvent kinetic isotope effect and lack of viscosity effects support that a step coupled to FMN regeneration determines the rate of the reaction. Our work dissects the complex mechanistic and structural features of this dehydrogenation reaction. This sets the stage to utilizing NdasCDO as a biocatalyst and expands the FMN-dependent oxidase family to include enzyme filaments.

biochemistry↗

Distinct haplogroups with star-like diversification in the APOBEC3 regulatory region indicate ancient viral pandemics before and during the Out-of-Africa migration of modern humans

Human anti-viral APOBEC3s are crucial components of the innate immune response, playing a key role in inhibiting viral replication and proliferation by inducing mutations in viral genomes. In this study, in a regulatory region of [~]16 kb in the APOBEC3 gene cluster on human chromosome 22, we identified three distinct haplogroups that are maintained at high frequencies in both African and non-African populations today. Despite the long persistence of these haplogroups, one of which is shared by archaic hominins, the nucleotide diversity within each haplogroup was unusually low in non-Africans. The simultaneous observations of low intra-haplogroup diversity and high inter-haplogroup divergence were incompatible with neutrality, as demonstrated by the genome-wide empirical distribution and coalescence-based simulation tests. In addition to the three shared haplogroups, an ancestral sequence group was identified exclusively in African populations. To explain these features and investigate the underlying evolutionary mechanisms, we performed forward simulations to model the joint effects of balancing selection and population bottlenecks. We demonstrated the operation of balancing selection over the past [~]1 million years, as well as a hitherto unexplored reduction in intra-haplogroup diversity. The only unexplainable observation was the low diversity within African haplogroup I, which is unaffected by the non-African-specific bottleneck. We hypothesized that an extra haplotype turnover within haplogroup I occurred prior to the Out-of-Africa dispersal of modern humans. Finally, we discussed the functional importance of the APOBEC3 regulatory region in terms of evolutionary conservation in Hominidae, a target of natural selection in modern humans, and its relevance in GTEx eQTL. Significance statementThis study provides new insights into the joint effects of population bottlenecks and balancing selection on genetic diversity within haplogroups. First, we reveal how balancing selection interacts with demographic history to shape intra-haplogroup diversity, a previously unexplored aspect. Second, our findings are generalizable, as illustrated by the ABO locus--an established example of balancing selection--where low within-haplogroup diversity and high between-haplogroup divergence are also observed in non-African populations. Third, the involvement of balancing selection in a highly conserved regulatory region suggests functional significance in modulating the expression of all seven APOBEC3 genes. While further validation is needed, our findings underscore the evolutionary and functional importance of this region in human innate immunity.

evolutionary biology↗

Transcription promotes the restoration of chromatin following DNA replication.

DNA replication results in the transient eviction of nucleosomes, RNAPII and transcription regulators. How chromatin organization is duplicated on the two daughter strands is a central question in epigenetics. In mammals, transcription restarts on newly replicated DNA within a couple of hours, promoting chromatin accessibility. However, the role of transcription in the restoration of other chromatin determinants following DNA replication remains unclear. Here we have monitored protein re-association to newly replicated DNA upon inhibition of transcription using iPOND coupled to quantitative mass spectrometry. We show that nucleosome assembly and the re-establishment of most histone modifications are uncoupled from transcription restart. However, upon transcription inhibition, the re-association of many proteins was altered, including ATP-dependent remodellers, transcription regulators, the histone variant H2A.Z, histone modifiers as well as the restoration of H3.3K36me2. Finally, transcription also provoked the recruitment of several DNA repair proteins, revealing that transcription promotes chromatin reestablishment post-replication but is also a potential source of genotoxic stress.

molecular biology↗

An intramolecular bivalent degrader glues an intrinsic BRD4-DCAF16 interaction

Targeted protein degradation is a pharmacological modality based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal degradation. This has been achieved either via bifunctional compounds (PROTACs) composed of two separate warheads that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target1-4. Using orthogonal genetic screening, biophysical characterization, and structural reconstitution, we investigate the mode of action of bifunctional BRD2/4 degraders (IBG1-4) and find that - instead of connecting target and ligase in trans as PROTACs do - they simultaneously engage two adjacent domains of the target protein in cis. This conformational change glues BRD4 to the E3 ligases DCAF11 or DCAF16, leveraging intrinsic target-ligase affinities which, albeit pre-existing, do not translate to BRD4 degradation in absence of compound. Structural insights into the ternary BRD4:IBG1:DCAF16 complex guided the rational design of improved degraders of low picomolar potency. We thus introduce a new modality in targeted protein degradation, termed intramolecular bivalent glues (IBGs), which work by bridging protein domains to enhance surface complementarity with E3 ligases for productive ubiquitination and degradation.

biochemistry↗

Structure and mechanism of the type I-G CRISPR effector

Type I CRISPR systems are the most common CRISPR type found in bacteria. They use a multisubunit effector, guided by crRNA, to detect and bind dsDNA targets, forming an R-loop and recruiting the Cas3 enzyme to facilitate target DNA destruction, thus providing immunity against mobile genetic elements. Subtypes have been classified into families A-G, with type I-G being the least well understood. Here, we report the composition, structure and function of the type I-G Cascade CRISPR effector from Thioalkalivibrio sulfidiphilus, revealing key new molecular details. The unique Csb2 subunit processes pre-crRNA, remaining bound to the 3 end of the mature crRNA, and seven Cas7 subunits form the backbone of the effector. Cas3 associates stably with the effector complex via the Cas8g subunit and is important for target DNA recognition. Structural analysis by cryo-Electron Microscopy reveals a strikingly curved backbone conformation with Cas8g spanning the belly of the structure. Type I-G Cascade is one of the most streamlined Class 1 CRISPR effectors. These biochemical and structural insights shed new light on the diversity of type I systems and open the way to applications in genome engineering.

microbiology↗

Non-canonical scaffold-type ligase complex mediates protein UFMylation

Protein UFMylation is emerging as a posttranslational modification essential for endoplasmic reticulum and cellular homeostasis. Despite its biological importance, we have a poor understanding of how UFM1 is conjugated onto substrates. Here, we use a rebuilding approach to define the minimal requirements of protein UFMylation. We find that the reported E3 ligase UFL1 is inactive on its own and identify UFBP1 to bind UFL1 to form an active E3 ligase complex. While UFC1 is an intrinsically Cys-reactive E2, we do not identify any catalytic cysteines on UFL1/UFBP1, suggesting a scaffold-type E3 ligase mechanism. Interestingly, the E3 ligase complex consists of winged-helix (WH) domain repeats that activate UFC1 for aminolysis. We identify the adaptor protein CDK5RAP3 to bind to and regulate E3 ligase activity potentially by preventing off-target UFMylation. In summary, our work identifies the minimal requirements for UFMylation and reveals regulatory principles of this atypical E3 ligase complex.

biochemistry↗

Structure of the chromatin remodelling enzyme Chd1 bound to a ubiquitinylated nucleosome

ATP-dependent chromatin remodelling proteins represent a diverse family of proteins that share ATPase domains that are adapted to regulate protein-DNA interactions. Here we present structures of the yeast Chd1 protein engaged with nucleosomes in the presence of the transition state mimic ADP-beryllium fluoride. The path of DNA strands through the ATPase domains indicates the presence of contacts conserved with single strand translocases and additional contacts with both strands that are unique to Snf2 related proteins. The structure provides connectivity between rearrangement of ATPase lobes to a closed, nucleotide bound state and the sensing of linker DNA. Two turns of linker DNA are prised off the surface of the histone octamer as a result of Chd1 binding, and both the histone H3 tail and ubiquitin conjugated to lysine 120 are re-orientated towards the unravelled DNA. This indicates how changes to nucleosome structure can alter the way in which histone epitopes are presented.

molecular biology↗

SINHCAF/FAM60A links SIN3A function to the hypoxia response and its levels are predictive of cancer patient survival

The SIN3A-HDAC complex is a master transcriptional repressor, required for development but often deregulated in disease. Here, we report that the recently identified new component of this complex, SINHCAF/FAM60A, links the SIN3A-HDAC co-repressor complex function to the hypoxia response. SINHCAF Chromatin Immunoprecipitation-sequencing and gene expression analysis reveal a signature associated with the activation of the hypoxia response. We show that SINHCAF specifically repress HIF 2 mRNA and protein expression resulting in functional cellular changes in in-vitro angiogenesis, and proliferation. Analysis of patient datasets demonstrates that SINHCAF and HIF 2 mRNA levels are inversely correlated and predict contrasting outcomes for patient survival in both colon and lung cancer. This relationship is also observed in a mouse model of colon cancer, indicating an evolutionary conserved mechanism. Our analysis reveals an unexpected link between SINHCAF and cancer cell signalling via regulation of the hypoxia response that is predictive of poor patient outcome.

molecular biology↗

Structural reorganization of the chromatin remodeling enzyme Chd1 upon engagement with nucleosomes.

The yeast Chd1 protein acts to position nucleosomes across genomes. Here we model the structure of the Chd1 protein in solution and when bound to nucleosomes. In the apo state the DNA binding domain contacts the edge of the nucleosome while in the presence of the non-hydrolyzable ATP analog, ADP-beryllium fluoride, we observe additional interactions between the ATPase domain and the adjacent DNA gyre 1.5 helical turns from the dyad axis of symmetry. Binding in this conformation involves unravelling the outer turn of nucleosomal DNA and requires substantial reorientation of the DNA binding domain with respect to the ATPase domains. The orientation of the DNA-binding domain is mediated by sequences in the N-terminus and mutations to this part of the protein have positive and negative effects on Chd1 activity. These observations indicate that the unfavourable alignment of C-terminal DNA binding region in solution contributes to an auto-inhibited state.

molecular biology↗