Biology subjects

Dunkle, J. A.

Publications and source records attributed to Dunkle, J. A..

5 recordsLinked to original sources

bioRxiv2025

Cas10 residues lining the target RNA binding channel regulate interference by distinguishing cognate target RNA from mismatched targets

Type III CRISPR systems are defined by the presence of the Cas10 protein and are among the most abundant CRISPR systems in nature. Cas10 forms a complex with crRNA and several Cas proteins that surveils bacterial cells for foreign RNA molecules and when they are detected it activates a cascade of interference activities. The synthesis of the cyclic oligoadenylate signaling molecule by Cas10 is a key aspect of the interference cascade. Despite structures of the Cas10 complex bound to target RNAs, the molecular mechanism by which Cas10 senses the bound state to license interference is lacking. We identified five residues in S. epidermidis Cas10, two in the Cas10 Palm2 domain and three in domain 4, that line the target RNA binding channel. We assessed the contribution of these residues to interference in the context of a cognate or mismatched target RNA. We found that the residues regulate whether a mismatched crRNA-target RNA duplex is able to activate interference in vivo. We purified two site-directed mutants of Cas10-Csm and show with in vitro cOA synthesis assays they demonstrate enhanced discrimination of cognate versus mismatched targets.

molecular biology↗

bioRxiv2024

Two conserved arginine residues facilitate C-S bond cleavage and persulfide transfer in Suf family cysteine desulfurases.

Under conditions of oxidative stress or iron starvation, iron-sulfur cluster biogenesis in E. coli is initiated by the cysteine desulfurase, SufS, via the SUF pathway. SufS is a type II cysteine desulfurase that catalyzes the PLP-dependent breakage of an L-cysteine C-S bond to generate L-alanine and a covalent active site persulfide as products. The persulfide is transferred from SufS to SufE and then to the SufBC2D complex, which utilizes it in iron-sulfur cluster biogenesis. Several lines of evidence suggest two conserved arginine residues that line the solvent side of the SufS active site could be important for function. To investigate the mechanistic roles of R56 and R359, the residues were substituted using site-directed mutagenesis to obtain R56A/K and R359A/K SufS variants. Steady state kinetics indicated R56 and R359 have moderate defects in the desulfurase half reaction but major defects in the transpersulfurase step. Fluorescence polarization binding assays showed that the loss of activity was not due to a defect in forming the SufS/SufE complex. Structural characterization of R56A SufS shows loss of electron density for the 3-4 loop at the R56/G57 positions, consistent with a requirement of R56 for proper loop conformation. The structure of R359A SufS exhibits a conformational change in the 3-4 loop allowing R56 to enter the active site and mimics the residues position in the PLP-cysteine aldimine structure. Taken together, the kinetic, binding, and structural data support a mechanism where R359 plays a role in linking SufS catalysis with modulation of the 3-4 loop to promote a close-approach interaction of SufS and SufE conducive to persulfide transfer.

biochemistry↗

bioRxiv2024

The structure of the SufS-SufE complex reveals interactions driving protected persulfide transfer in iron-sulfur cluster biogenesis

Fe-S clusters are critical cofactors for redox chemistry in all organisms. The cysteine desulfurase, SufS, provides sulfur in the SUF Fe-S cluster bioassembly pathway. SufS is a dimeric, PLP-dependent enzyme that uses cysteine as a substrate to generate alanine and a covalent persulfide on an active site cysteine residue. SufS enzymes are activated by an accessory transpersulfurase protein, either SufE or SufU depending on the organism, which accepts the persulfide product and delivers it to downstream partners for Fe-S assembly. Here, using E. coli proteins, we present the first X-ray crystal structure of a SufS/SufE complex. There is a 1:1 stoichiometry with each monomeric unit of the EcSufS dimer bound to one EcSufE subunit, though one EcSufE is rotated [~]7{degrees} closer to the EcSufS active site. EcSufE makes clear interactions with the 16 helix of EcSufS and site-directed mutants of several 16 residues were deficient in EcSufE binding. Analysis of the EcSufE structure showed a loss of electron density at the EcSufS/EcSufE interface for a flexible loop containing the highly conserved residue R119. An R119A EcSufE variant binds EcSufS but is not active in cysteine desulfurase assays and fails to support Fe-S cluster bioassembly in vivo. 35S-transfer assays suggest that R119A EcSufE can receive a persulfide, suggesting the residue may function in a release mechanism. The structure of the EcSufS/EcSufE complex allows for comparison with other cysteine desulfurases to understand mechanisms of protected persulfide transfer across protein interfaces.

biochemistry↗

bioRxiv2024

Two dynamic, N-terminal regions are required for function in Ribosomal RNA Adenine Dimethylase family members

The Ribosomal RNA Adenine Dimethylase (RRAD) family of enzymes facilitate ribosome maturation in all organisms by dimethylating two nucleotides of small subunit rRNA. Prominent members of this family are the human DIMT1 and bacterial KsgA enzymes. A sub-group of RRAD enzymes, named erythromycin resistance methyltransferases (Erm) dimethylate a specific nucleotide in large subunit rRNA to confer antibiotic resistance. How these enzymes regulate methylation so that it only occurs on the specific substrate is not fully understood. While performing random mutagenesis on the catalytic domain of ErmE, we discovered that mutants in an N-terminal region of the protein that is disordered in the ErmE crystal structure are associated with a loss of antibiotic resistance. By subjecting site-directed mutants of ErmE and KsgA to phenotypic and in vitro assays we found that the N-terminal region is critical for activity in RRAD enzymes: the N-terminal basic region promotes rRNA binding and the conserved motif likely assists in juxtaposing the adenosine substrate and the SAM cofactor. Our results and emerging structural data suggest this dynamic, N-terminal region of RRAD enzymes becomes ordered upon rRNA binding forming a cap on the active site required for methylation.

biochemistry↗

bioRxiv2022

The structure of a Type III-A CRISPR-Cas effector complex reveals conserved and idiosyncratic contacts to target RNA and crRNA among Type III-A systems

Type III CRISPR-Cas systems employ multiprotein effector complexes bound to small CRISPR RNAs (crRNAs) to detect foreign RNA transcripts and elicit a complex immune response that leads to the destruction of invading RNA and DNA. Type III systems are among the most widespread in nature, and emerging interest in harnessing these systems for biotechnology applications highlights the need for detailed structural analyses of representatives from diverse organisms. We performed cryo-EM reconstructions of the Type III-A Cas10-Csm effector complex from S. epidermidis bound to an intact, cognate target RNA and identified two oligomeric states, a 276 kDa complex and a 318 kDa complex. 3.1 [A] density for the well-ordered 276 kDa complex allowed construction of atomic models for the Csm2, Csm3, Csm4 and Csm5 subunits within the complex along with the crRNA and target RNA. We also collected small-angle X-ray scattering data which was consistent with the 276 kDa Cas10-Csm architecture we identified. Detailed comparisons between the S. epidermidis Cas10-Csm structure and the well-resolved bacterial (S. thermophilus) and archaeal (T. onnurineus) Cas10-Csm structures reveal differences in how the complexes interact with target RNA and crRNA which are likely to have functional ramifications. These structural comparisons shed light on the unique features of Type III-A systems from diverse organisms and will assist in improving biotechnologies derived from Type III-A effector complexes.

molecular biology↗