Biology subjects

Chait, B.

Publications and source records attributed to Chait, B..

9 recordsLinked to original sources

bioRxiv2025

Structural basis for Lamassu-based antiviral immunity and its evolution from DNA repair machinery

Bacterial immune systems exhibit remarkable diversity and modularity, as a consequence of the continuous selective pressures imposed by phage predation. Despite recent mechanistic advances, the evolutionary origins of many antiphage immune systems remain elusive, especially for those that encode homologs of the Structural Maintenance of Chromosomes (SMC) superfamily, which are essential for chromosome maintenance and DNA repair across domains of life. Here, we elucidate the structural basis and evolutionary emergence of Lamassu, a bacterial immune system family featuring diverse effectors but a core conserved SMC-like sensor. Using cryo-EM, we determined structures of the Vibrio cholerae Lamassu complex in both apo- and dsDNA-bound states, revealing unexpected stoichiometry and topological architectures. We further demonstrate how Lamassu specifically senses dsDNA in vitro and phage replication origins in vivo, thereby triggering the formation of LmuA tetramers that activate the Cap4 nuclease domain. Our findings reveal that Lamassu evolved via exaptation of the bacterial Rad50-Mre11 DNA repair system to form a compact, modular sensor for viral replication, exemplifying how cellular machinery can be co-opted for novel immune functions.

microbiology↗

bioRxiv2024

DnaB and DciA: Mechanisms of Helicase Loading and Translocation on ssDNA

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/622779v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@b0641dorg.highwire.dtl.DTLVardef@7caf59org.highwire.dtl.DTLVardef@1dc0fccorg.highwire.dtl.DTLVardef@913ce2_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO The Vibrio cholerae (Vc) DnaB replicative helicase structure bound to single-stranded (ss) DNA is depicted in the ribbon (top left) and sphere-cylinder representation (top right). In the bottom center is a native mass spectrum showing Vc DnaB helicase loading onto single-stranded DNA (ssDNA). C_FIG Replicative helicases are assembled on chromosomes by helicase loaders before initiation of DNA replication. Here, we investigate mechanisms used by the bacterial Vibrio cholerae (Vc) DnaB replicative helicase and the DciA helicase loader. Structural analysis of the ATP{gamma}S form of the VcDnaB-ssDNA complex reveals a configuration distinct from that seen with GDP*AlF4. With ATP{gamma}S, the amino-terminal (NTD) tier, previously found as an open spiral in the GDP*AlF4 complex, adopts a closed planar arrangement. Further, the DnaB subunit at the top of the carboxy-terminal spiral (CTD) tier is displaced by [~]25 [A] between the two forms. We suggest that remodeling the NTD layer between closed planar and open spiral configurations and migration of two distinct CTDs to the top of the DnaB spiral, repeated three times, mediates hand-over-hand translocation. Biochemical analysis suggests that VcDciA leverages its Lasso domain to contact DnaB near its Docking-Linker-Helix interface. Up to three copies of VcDciA bind to VcDnaB and suppress its ATPase activity during loading onto physiological DNA substrates. Our data suggest that DciA loads DnaB onto DNA using the ring-opening mechanism.

biochemistry↗

bioRxiv2024

Distinct modes of coupling between VCP, an essential unfoldase, and deubiquitinases

Errors in proteostasis, which requires regulated degradation and recycling of diverse proteins, are linked to aging, cancer and neurodegenerative disease (1). In particular, recycling proteins from multiprotein complexes, organelles and membranes is initiated by ubiquitylation, extraction and unfolding by the essential mechanoenzyme VCP (2-4), and ubiquitin removal by deubiquitinases (DUBs), a class of [~]100 ubiquitin-specific proteases in humans (5, 6). As VCPs substrate recognition requires ubiquitylation, the removal of ubiquitins from substrates for recycling must follow extraction and unfolding. How the activities of VCP and different DUBs are coordinated for protein recycling or other fates is unclear. Here, we employ a photochemistry-based approach to profile proteome-wide domain-specific VCP interactions in living cells (7). We identify DUBs that bind near the entry, exit, or both sites of VCPs central pore, the channel for ATP-dependent substrate translocation (8-10). From this set of DUBs, we focus on VCPIP1, required for organelle assembly and DNA repair (11-13), that our chemical proteomics workflow indicates binds the central pores entry and exit sites. We determine a [~]3[A] cryo-EM structure of the VCP-VCPIP1 complex and find up to 3 VCPIP1 protomers interact with the VCP hexamer. VCPIP1s UBX-L domain binds VCPs N-domain in a down conformation, linked to VCPs ADP-bound state (2, 14), and the deubiquitinase domain is positioned at the central pores exit site, poised to remove ubiquitin following substrate unfolding. We find that VCP stimulates VCPIP1s DUB activity and use mutagenesis and single-molecule mass photometry assays to test the structural model. Together, our data suggest that DUBs bind VCP at distinct sites and reveal how the two enzyme activities can be coordinated to achieve specific downstream outcomes for ubiquitylated proteins.

biochemistry↗

bioRxiv2024

Exportin-1 functions as an adaptor for transcription factor-mediated docking of chromatin at the nuclear pore complex.

Nuclear pore proteins (Nups) in yeast, flies and mammals physically interact with hundreds or thousands of chromosomal sites, which impacts transcriptional regulation. In budding yeast, transcription factors mediate interaction of Nups with enhancers of highly active genes. To define the molecular basis of this mechanism, we exploited a separation-of-function mutation in the Gcn4 transcription factor that blocks its interaction with the nuclear pore complex (NPC) without altering its DNA binding or activation domains. SILAC mass spectrometry revealed that this mutation reduces the interaction of Gcn4 with the highly conserved nuclear export factor Crm1/Xpo1. Crm1 both interacts with the same sites as Nups genome-wide and is required for Nup2 to interact with the yeast genome. In vivo, Crm1 undergoes extensive and stable interactions with the NPC. In vitro, Crm1 binds to Gcn4 and these proteins form a complex with the nuclear pore protein Nup2. Importantly, the interaction between Crm1 and Gcn4 does not require Ran-GTP, suggesting that it is not through the nuclear export sequence binding site. Finally, Crm1 stimulates DNA binding by Gcn4, supporting a model in which allosteric coupling between Crm1 binding and DNA binding permits docking of transcription factor-bound enhancers at the NPC.

cell biology↗

bioRxiv2023

Structural and functional insights into the enzymatic plasticity of the SARS-CoV-2 NiRAN Domain

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.

biophysics↗

bioRxiv2022

An Autoinhibited Conformation of the DnaB-Replicative Helicase phage Lambda P Loader Complex

Replicative helicases require loader proteins for assembly at the origins of DNA replication. Multiple copies of the bacteriophage {lambda}P (P) loader bind to and load the E. coli DnaB (B) replicative helicase on replication-origin-derived single-stranded DNA. We find that the E. coli DnaB*{lambda}P complex exists in two forms: B6P5 and B6P6. In the 2.66 [A] cryo-EM model of B6P5, five copies of the {lambda}P loader assemble into a crown-like shape that tightly grips DnaB. In this complex, closed planar DnaB is reconfigured into an open spiral with a sufficiently sized breach to permit ssDNA to enter an internal chamber. The transition to the open spiral involves {lambda}P-mediated changes to the Docking Helix (DH)-Linker Helix (LH) interface. The loader directly stabilizes the open spiral. Unexpectedly, one {lambda}P chain in B6P5 is bound across the breach, precluding entry of replication-origin-derived ssDNA into DnaBs central chamber. We suggest that the B6P6 complex is an early intermediate in the helicase activation pathway wherein neither the DnaB helicase nor the {lambda}P loader has attained its final form. DnaB in this complex adopts a partially open planar configuration, termed ajar planar. The partially ordered {lambda}P loader assembly features a much looser interaction with DnaB. The ssDNA and ATP sites in both complexes are in a configuration ill-suited for binding or hydrolysis. Our work specifies the conformational changes required for the intermediate B6P6 to transition to B6P5 on the pathway to recruitment by the initiator protein complex to the replication origin. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/522210v4_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1c44809org.highwire.dtl.DTLVardef@1554deeorg.highwire.dtl.DTLVardef@16f6df9org.highwire.dtl.DTLVardef@9d12dd_HPS_FORMAT_FIGEXP M_FIG C_FIG The DnaB helicase loading pathway at the phage {lambda} replication origin populates two intermediate states, distinguished by the number of {lambda}P loaders present. The DnaB ring in the B6P6 complex is planar and partially open. Although it binds six copies of the {lambda}P loader, the ajar planar state of DnaB yields inchoate interactions with the loader. During the maturation of the complex, the planar state of DnaB is reconfigured into an open spiral in the B6P5 complex, which the pentameric {lambda}P ensemble now grips tightly. This transition required the eviction of one copy of the loader. Although the breach in the DnaB open spiral is sufficiently sized for entry of ssDNA into the internal chamber, the disposition of one {lambda}P chain loader across the single breached interface in DnaB effectively blocks the path to physiological replication origin-derived ssDNA. DnaB is depicted in white/gray ribbon format under a transparent surface. The {lambda}P chains are colored orange and shades of blue. The pink cylinder represents the expected path of ssDNA through DnaB.

biochemistry↗

bioRxiv2021

Comprehensive Structure and Functional Adaptations of the Yeast Nuclear Pore Complex

Nuclear Pore Complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the yeast NPC in which the inner ring is resolved by cryo-EM at - helical resolution to show how flexible connectors tie together different structural and functional layers in the spoke. These connectors are targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and karyopherins have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We also provide evidence for three major NPC variants that foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies to provide a comprehensive model of the in situ NPC with a radially-expanded inner ring. Our model reveals novel features of the central transporter and nuclear basket, suggests a role for the lumenal ring in restricting dilation and highlights the structural plasticity required for transport by the NPC.

biophysics↗

bioRxiv2021

Immunodominant surface epitopes power immune evasion in the African trypanosome

The African trypanosome survives the immune response of its mammalian host by antigenic variation of its major surface antigen (the Variable Surface Glycoprotein, or VSG). Here we describe the antibody repertoires elicited by different VSGs. We show that the repertoires are highly restricted, and are directed predominantly to distinct epitopes on the surface of the VSGs. They are also highly discriminatory: minor alterations within these exposed epitopes confer antigenically-distinct properties to these VSGs and elicit different repertoires. We propose that the patterned and repetitive nature of the VSG coat focuses host immunity to a restricted set of immunodominant epitopes per VSG, eliciting a highly stereotyped response, minimizing cross reactivity between different VSGs and facilitating prolonged immune evasion through epitope variation.

immunology↗

bioRxiv2021

Analysis of the Mechanosensor Channel Functionality of TACAN

Mechanosensitive ion channels mediate transmembrane ion currents activated by mechanical forces. A mechanosensitive ion channel called TACAN was recently reported. We began to study TACAN with the intent to understand how it senses mechanical forces and functions as an ion channel. Using cellular patch-recording methods we failed to identify mechanosensitive ion channel activity. Using membrane reconstitution methods we found that TACAN, at high protein concentrations, produces non-selective, heterogeneous conduction levels that are not mechanosensitive and are most consistent with disruptions of the lipid bilayer. We determined the structure of TACAN using single particle cryo-EM and observe that it forms a symmetrical dimeric transmembrane protein. Each protomer contains an intracellular-facing cleft with a coenzyme-A co-factor, confirmed by mass spectrometry. The TACAN protomers are related in 3-dimensional structure to a fatty acid elongase, ELOVL. Whilst its physiological function remains unclear, we anticipate that TACAN is not a mechanosensitive ion channel.

biophysics↗