Browsing by Author "Nasef, Mohamed"

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    The beta-latch structural element of the SufS cysteine desulfurase mediates active site accessibility and SufE transpersulfurase positioning
    (Elsevier, 2023) Gogar, Rajleen K.; Carroll, Franki; Conte, Juliana, V; Nasef, Mohamed; Dunkle, Jack A.; Frantom, Patrick A.; University of Alabama Tuscaloosa
    Under oxidative stress and iron starvation conditions, Escherichia coli uses the Suf pathway to assemble iron-sulfur clusters. The Suf pathway mobilizes sulfur via SufS, a type II cysteine desulfurase. SufS is a pyridoxal-5'-phosphate-depen-dent enzyme that uses cysteine to generate alanine and an active-site persulfide (C364-S-S-). The SufS persulfide is pro-tected from external oxidants/reductants and requires the transpersulfurase, SufE, to accept the persulfide to complete the SufS catalytic cycle. Recent reports on SufS identified a conserved "11-latch" structural element that includes the alpha 6 helix, a glycine-rich loop, a 11-hairpin, and a cis-proline residue. To identify a functional role for the 11-latch, we used site -directed mutagenesis to obtain the N99D and N99A SufS var-iants. N99 is a conserved residue that connects the alpha 6 helix to the backbone of the glycine-rich loop via hydrogen bonds. Our x-ray crystal structures for N99A and N99D SufS show a dis-torted beta-hairpin and glycine-rich loop, respectively, along with changes in the dimer geometry. The structural disruption of the N99 variants allowed the external reductant TCEP to react with the active-site C364-persulfide intermediate to complete the SufS catalytic cycle in the absence of SufE. The substitutions also appear to disrupt formation of a high -affinity, close approach SufS-SufE complex as measured with fluorescence polarization. Collectively, these findings demon-strate that the 11-latch does not affect the chemistry of persul-fide formation but does protect it from undesired reductants. The data also indicate the 11-latch plays an unexpected role in forming a close approach SufS-SufE complex to promote persulfide transfer.
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    The effect of crRNA-target mismatches on cOA-mediated interference by a type III-A CRISPR-Cas system
    (Taylor & Francis, 2022) Nasef, Mohamed; Khweis, Sarah A. A.; Dunkle, Jack A. A.; University of Alabama Tuscaloosa
    CRISPR systems elicit interference when a foreign nucleic acid is detected by its ability to base-pair to crRNA. Understanding what degree of complementarity between a foreign nucleic acid and crRNA is required for interference is a central question in the study of CRISPR systems. A clear description of which target-crRNA mismatches abrogate interference in type III, Cas10-containing, CRISPR systems has proved elusive due to the complexity of the system which utilizes three distinct interference activities. We characterized the effect of target-crRNA mismatches on in vitro cyclic oligoadenylate (cOA) synthesis and in vivo in an interference assay that depends on cOA synthesis. We found that sequence context affected whether a mismatched target was recognized by crRNA both in vitro and in vivo. We also investigated how the position of a mismatch within the target-crRNA duplex affected recognition by crRNA. Our data provide support for the hypothesis that a Cas10-activating region exists in the crRNA-target duplex, that the Cas10-proximal region of the duplex is the most critical in regulating cOA synthesis. Understanding the rules governing target recognition by type III CRISPR systems is critical: as one of the most prevalent CRISPR systems in nature, it plays an important role in the survival of many genera of bacteria. Recently, type III systems were re-purposed as a sensitive and accurate molecular diagnostic tool. Understanding the rules of target recognition in this system will be critical as it is engineered for biotechnology purposes.
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    Investigation of the RNA-Protein Structure-Function Relationships in the CRISPR-Cas10 Complex and Erythromycin-Resistance RNA Methyltransferases
    (University of Alabama Libraries, 2021) Nasef, Mohamed; Dunkle, Jack A.; University of Alabama Tuscaloosa
    CRISPR-Cas systems are a class of adaptive immune systems in prokaryotes that use small CRISPR RNAs (crRNAs) in conjunction with CRISPR-associated (Cas) nucleases to recognize and degrade foreign nucleic acids. Recent studies have revealed that type III CRISPR-Cas systems synthesize second messenger molecules, previously unknown to exist in prokaryotes, known as cyclic oligoadenylates (cOA). These molecules activate the Csm6 nuclease to promote RNA degradation and may also coordinate additional cellular responses to foreign nucleic acids. Although cOA production has been reconstituted and characterized for a few bacterial and archaeal type III systems, cOA generation and its regulation have not been explored for the Gram-positive bacterium, Staphylococcus epidermidis, as well as the specific target RNA sequence requirements. In chapter 2, we demonstrate that this system performs Mg2+ dependent synthesis of 3-6 nt cOA. We show that activation of cOA synthesis is perturbed by single nucleotide mismatches between the crRNA and target RNA at discrete positions and synthesis is antagonized by Csm3-mediated target RNA cleavage. In chapter 3, we mechanistically investigated the effect of single and multiple mismatches on the activation of cOA synthesis of type III-A Cas10-Csm complex from S. epidermidis. We show that mismatches at specific positions in target RNA significantly reduced the amount of cOAs produced in a sequence context-dependent manner. We also show that the defects are not due to perturbations in binding efficiencies or in RNA cleavage.In chapter 4, we focus on an erythromycin resistance methyltransferases (Erm) found in many Gram-positive pathogens. Erm proteins are S-adenosyl methionine-dependent Rossmann-fold methyltransferases which convert A2058 of 23S rRNA to m62A2058. This modificationsterically blocks binding of several classes of antibiotics to 23S rRNA, resulting in a multi-drug resistant phenotype in bacteria expressing the enzyme. ErmC is an erythromycin resistance methyltransferase found in many Gram-positive pathogens, while ErmE is found in the soil bacterium that biosynthesizes erythromycin. Whether ErmC and ErmE, which possess only 24 % sequence identity, use similar structural elements for rRNA substrate recognition and positioning is not known. To investigate this question, we used structural data from related proteins to guide site-saturation mutagenesis of key residues and characterized selected variants by antibiotic susceptibility testing, single turnover kinetics, and RNA affinity binding assays. We demonstrate that residues in α4, α5 and the α5-α6 linker are essential for methyltransferase function including: an aromatic residue on α4 that likely forms stacking interactions with the substrate adenosine, and basic residues in α5 and the α5-α6 linker which likely mediate conformational rearrangements in the protein and cognate rRNA upon interaction. The functional studies led us to a new structural model for the ErmC or ErmE-rRNA complex.
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    Regulation of cyclic oligoadenylate synthesis by the Staphylococcus epidermidis Cas10-Csm complex
    (Cold Spring Harbor Lab Press, 2019) Nasef, Mohamed; Muffly, Mary C.; Beckman, Andrew B.; Rowe, Sebastian J.; Walker, Forrest C.; Hatoum-Aslan, Asma; Dunkle, Jack A.; University of Alabama Tuscaloosa
    CRISPR-Cas systems are a class of adaptive immune systems in prokaryotes that use small CRISPR RNAs (crRNAs) in conjunction with CRISPR-associated (Cas) nucleases to recognize and degrade foreign nucleic acids. Recent studies have revealed that Type III CRISPR-Cas systems synthesize second messenger molecules previously unknown to exist in prokaryotes, cyclic oligoadenylates (cOA). These molecules activate the Csm6 nuclease to promote RNA degradation and may also coordinate additional cellular responses to foreign nucleic acids. Although cOA production has been reconstituted and characterized for a few bacterial and archaeal Type III systems, cOA generation and its regulation have not been explored for the Staphylococcus epidermidis Type III-A CRISPR-Cas system, a longstanding model for CRISPR-Cas function. Here, we demonstrate that this system performs Mg2+-dependent synthesis of 3-6 nt cOA. We show that activation of cOA synthesis is perturbed by single nucleotide mismatches between the crRNA and target RNA at discrete positions, and that synthesis is antagonized by Csm3-mediated target RNA cleavage. Altogether, our results establish the requirements for cOA production in a model Type III CRISPR-Cas system and suggest a natural mechanism to dampen immunity once the foreign RNA is destroyed.
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    Shared requirements for key residues in the antibiotic resistance enzymes ErmC and ErmE suggest a common mode of RNA recognition
    (Elsevier, 2020) Rowe, Sebastian J.; Mecaskey, Ryan J.; Nasef, Mohamed; Talton, Rachel C.; Sharkey, Rory E.; Halliday, Joshua C.; Dunkle, Jack A.; University of Alabama Tuscaloosa
    Erythromycin-resistance methyltransferases are SAM dependent Rossmann fold methyltransferases that convert A2058 of 23S rRNA to m(6) (2)A2058. This modification sterically blocks binding of several classes of antibiotics to 23S rRNA, resulting in a multidrug-resistant phenotype in bacteria expressing the enzyme. ErmC is an erythromycin resistance methyltransferase found in many Gram-positive pathogens, whereas ErmE is found in the soil bacterium that biosynthesizes erythromycin. Whether ErmC and ErmE, which possess only 24% sequence identity, use similar structural elements for rRNA substrate recognition and positioning is not known. To investigate this question, we used structural data from related proteins to guide site-saturation mutagenesis of key residues and characterized selected variants by antibiotic susceptibility testing, single turnover kinetics, and RNA affinity-binding assays. We demonstrate that residues in alpha 4, alpha 5, and the alpha 5-alpha 6 linker are essential for methyltransferase function, including an aromatic residue on alpha 4 that likely forms stacking interactions with the substrate adenosine and basic residues in alpha 5 and the alpha 5-alpha 6 linker that likely mediate conformational rearrangements in the protein and cognate rRNA upon interaction. The functional studies led us to a new structural model for the ErmC or ErmE-rRNA complex.
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    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
    (PLOS, 2023) Paraan, Mohammadreza; Nasef, Mohamed; Chou-Zheng, Lucy; Khweis, Sarah A. A.; Schoeffler, Allyn J. J.; Hatoum-Aslan, Asma; Stagg, Scott M. M.; Dunkle, Jack A. A.; University of Alabama Tuscaloosa; University of Illinois Urbana-Champaign; Loyola University New Orleans; Florida State University
    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 & ANGS; 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.