Biology subjects

Zimanyi, C. M.

Publications and source records attributed to Zimanyi, C. M..

4 recordsLinked to original sources

bioRxiv2025

Structures reveal how the Cas1-2/3 integrase captures, delivers, and integrates foreign DNA into CRISPR loci

Cas1 and Cas2 are the hallmark proteins of prokaryotic adaptive immunity. However, these two proteins are often fused to other proteins and the functional association of these fusions often remain poorly understood. Here we purify Cas1 and the Cas2/3 fusion protein from Pseudomonas aeruginosa. We determine multiple structures of the Cas1-2/3 complex at distinct stages of CRISPR adaptation. Collectively, these structures reveal a prominent, positively charged channel on one face of the integration complex that captures short fragments of foreign DNA. Foreign DNA binding triggers conformational changes in Cas2/3 that expose new DNA binding surfaces necessary for homing the DNA-bound integrase to specific CRISPR loci. The length of the foreign DNA substrate determines if Cas1-2/3 docks completely onto the CRISPR repeat to successfully catalyze two sequential transesterification reactions required for integration. Taken together, these structures clarify how the Cas1-2/3 proteins orchestrate foreign DNA capture, site-specific delivery, and integration of new DNA into the bacterial genome. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/658980v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@11ad39borg.highwire.dtl.DTLVardef@3a8474org.highwire.dtl.DTLVardef@1cdf25borg.highwire.dtl.DTLVardef@1e24ff5_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTS- A positively charged channel on the Cas1-2/3 complex captures fragments of DNA - A loop in the RecA1 domain controls access to the Cas3 nuclease active site - Foreign DNA binding allosterically regulates access to additional DNA binding sites - Distortion of the CRISPR repeat sequence licenses complete foreign DNA integration

molecular biology↗

bioRxiv2021

How ATP and dATP act as molecular switches to regulate enzymatic activity in the prototypic bacterial class Ia ribonucleotide reductase

Class Ia ribonucleotide reductases (RNRs) are subject to allosteric regulation to maintain the appropriate deoxyribonucleotide levels for accurate DNA biosynthesis and repair. RNR activity requires a precise alignment of its 2 and {beta}2 subunits such that a catalytically-essential radical species is transferred from {beta}2 to 2. In E. coli, when too many deoxyribonucleotides are produced, dATP binding to RNR generates an inactive 4{beta}4 state in which {beta}2 and 2 are separated, preventing radical transfer. ATP binding breaks the -{beta} interface, freeing {beta}2 and restoring activity. Here we investigate the molecular basis for allosteric activity regulation in the prototypic E. coli class Ia RNR. Through the determination of six crystal structures we are able to establish how dATP binding creates a binding pocket for {beta} on that traps {beta}2 in the inactive 4{beta}4 state. These structural snapshots also reveal the numerous ATP-induced conformational rearrangements that are responsible for freeing {beta}2. We further discover, and validate through binding and mutagenesis studies, a previously unknown nucleotide binding site on the subunit that is crucial for the ability of ATP to dismantle the inactive 4{beta}4 state. These findings have implications for the design of allosteric inhibitors for bacterial RNRs.

biochemistry↗

bioRxiv2018

Proton co-transport and voltage dependence enforce unidirectional metal transport in an Nramp transporter

Secondary transporters harness electrochemical energy to move substrate through structurally-enforced co-substrate \"coupling\". We untangle the \"proton-metal coupling\" behavior by a Natural resistance-associated macrophage protein (Nramp) transporter into two distinct phenomena: {Delta}pH stimulation of metal transport and metal stimulation of proton co-transport. Surprisingly, metal type dictates co-transport stoichiometry, leading to manganese-proton symport but cadmium uniport. Additionally, the membrane potential affects both the kinetics and thermodynamics of metal transport. A conserved salt-bridge network near the metal-binding site imparts voltage dependence and enables proton co-transport, properties that allow this Nramp transporter to maximize metal uptake and prevent deleterious back-transport of acquired metals. We provide a new mechanistic model for Nramp metal-proton symport in which, in addition to substrate gradients determining directionality as in canonical secondary transport, synergy between protein structure and physiological voltage enforces unidirectional substrate movement. Our results illustrate a functional advantage that arises from deviations from the traditional model of symport.

biochemistry↗

bioRxiv2018

Structures in multiple conformations reveal distinct transition metal and proton pathways in an Nramp transporter

Nramp family transporters--expressed in organisms from bacteria to humans--enable uptake of essential divalent transition metals via an alternating-access mechanism that includes proton co-transport. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp at complementary stages of its transport cycle to provide a thorough description of the Nramp transport cycle by identifying the key intramolecular rearrangements and changes to the metal coordination sphere. Strikingly, while metal transport requires cycling from outward-to inward-open states, efficient proton transport still occurs in outward-locked (but not inward-locked) DraNramp. We propose a model in which metal and proton enter the transporter via the same external pathway to the binding site, but follow separate routes to the cytoplasm, thus resolving the electrostatic dilemma of using a cation co-substrate to drive a cation primary substrate. Our results illustrate the flexibility of the LeuT fold to support a broad range of co-substrate coupling and conformational change mechanisms.

biochemistry↗