Biology subjects

CRANE, B. R.

Publications and source records attributed to CRANE, B. R..

9 recordsLinked to original sources

bioRxiv2026

The bacterial swarming factor SwrD forms hexameric rings reminiscent of DNA binding proteins.

Prokaryotic swarming is a collective surface-associated behavior that supports rapid colonization, biofilm formation, and host interactions. Swarming requires enhanced flagellar motor (FM) torque generation under high-load conditions. In many bacteria, this adaptation is achieved through increased stator unit recruitment and coordinated transcriptional regulation of motility genes. Genetic studies have implicated several relatively poorly characterized proteins in swarming, including SwrD, a component of the fla-che operon whose loss impairs torque generation in Bacillus subtilis. Intriguingly, SwrD is conserved in spirochetes that experience high viscous loads but do not exhibit canonical swarming, suggesting a broader functional role in FM regulation. Here, we structurally and biophysically characterize SwrD from B. subtilis and the spirochete Borrelia burgdorferi. In both cases, we show that SwrD assembles into a hexameric ring-like structure featuring a highly charged, disordered peripheral tail. Whereas SwrD displays structural similarities to DNA-binding proteins, key DNA interaction motifs are not conserved in SwrD. Deletion of swrD in B. subtilis results in pronounced swarming defects and altered expression of fla-che operon transcripts. In silico interaction analyses further identify the stator protein MotA as a high-confidence SwrD interactor, but this interaction could not be validated biochemically. Conserved SwrD residues mediate subunit interactions or locate to the hexamer periphery where they may mediate functionally important interactions.

biochemistry↗

bioRxiv2026

An NO-binding Cache domain receptor interacts with a Ser/Thr kinase through a conserved HAMP domain interaction

Cache_heme domains are a family of bacterial heme c proteins that combine a conserved Cache fold with an -helical insertion containing a heme-binding CXXCH motif. We characterize an unusual Cache_heme-HAMP (CHH) receptor from Pseudomonas azotoformans with a periplasmic NO-binding Cache_heme domain, transmembrane region, and a cytoplasmic HAMP domain uncoupled from intrinsic enzymatic output. Genomic analysis reveals that CHH receptors are frequently co-localized with genes encoding Ser/Thr kinases (STPKs) that possess a catalytic domain similar to PknB, but lack peptidoglycan-binding PASTA domains. Using biochemical assays, biolayer interferometry, SEC-MALS, and cryo-electron microscopy, we show that the P. azotoformans CHH receptor binds its operon-associated kinase with nanomolar affinity via conserved C-terminal repeat modules in the kinase composed of two pseudo-symmetric -helical bundles. Cryo-EM structures demonstrate that this interaction orders the otherwise flexible HAMP domain, with the repeated helical domains from the kinase contacting each HAMP subunit symmetrically. Kinase-receptor binding is ATP-independent, but kinase phosphorylation of the receptor at a specific HAMP threonine residue is substantially enhanced when NO binds to the heme c sensor domain. Thus, NO-induced conformational changes that transverse the membrane either modulate substrate accessibility in the HAMP or alter kinase activity directly. These findings potentially define a mode of bacterial NO signaling in which a periplasmic heme c receptor couples ligand sensing to cytoplasmic phosphorylation via a physically associated Ser/Thr kinase, a mechanism distinct from that of cytoplasmic NO sensors. The conserved nature of the kinase recognition motif suggests broader relevance of CHH-STPK interactions across proteobacteria.

biochemistry↗

bioRxiv2026

Signaling mechanism of the transmembrane energy receptor Aer

The E. coli aerotaxis receptor Aer is a bacterial chemoreceptor that senses intracellular redox changes via an N terminal PAS domain bound to a flavin adenine dinucleotide (FAD) cofactor. Distinct from canonical methyl-accepting chemotaxis proteins (MCPs) such as Tar/Tsr, Aer lacks a periplasmic ligand-binding domain and adaptive methylation, transmitting conformational signals laterally from the PAS domain to the HAMP domain and the methylation helix-like cap (MHL cap) of the kinase control domain (KCD). To elucidate the Aer signalling mechanism, we determined cryo electron microscopy (cryo EM) structures of full length Aer in oxidized flavin quinone (kinase on) and reduced semiquinone (kinase off) states. Structural comparison reveals redox linked rearrangements of the FAD binding pocket, reorientation of PAS-HAMP interactions, and strikingly altered MHL cap stability. PAS-MHL-cap contact in the oxidized state compresses the receptor and stabilized proximal KCD helices, whereas reduction disrupts these contacts, increasing KCD flexibility. To probe distal effects on KCD architecture, we performed nanodisc reconstitution and pulse dipolar ESR spectroscopy on spin labelled positions along the four helix bundle. Distance distributions indicate redox dependent changes in helix separation, particularly at the C terminal MHL2 region, consistent with PAS driven loosening of KCD packing in kinase off states. These data support a model in which FAD redox chemistry reorganizes flavin pocket residues that in turn subtly alter PAS conformation to influence PAS-HAMP and PAS-MHL-cap packing and hence KCD conformational stability. The findings reveal an Aer specific signaling axis distinct from periplasmic ligand binding MCPs that has adapted MCP architecture for lateral PAS input and cytoplasmic redox sensing.

biochemistry↗

bioRxiv2026

Modulating radical propagation in proteins by proton-coupled electron transfer and hydrogen bonding

Long-range protein electron transfer (ET) often depends on tryptophan and tyrosine residues acting as radical relay sites. For example, cytochrome c peroxidase (CcP) generates a W191^dot+ radical to increase ET from cytochrome c (Cc) to the active center. W191 substitution to Tyr reduces ET rates, but introduction of an adjacent general base (as Glu or His) at position 232 (Y191:E/H232 CcP) recovers activity. E232 fluorination lowers the pKa of the conjugate base and confirms that a hydrogen bond is critical to elevate the Y191^dot formal potential for effective ET. Photoinitiated ET between Zn-porphyrin (ZnP) CcP (ZnCcP) and Cc also depends on activating Y191 with a basic residue, but through a different mechanism than for the peroxide-driven system. In ZnCcP, pH dependencies and solvent isotope effects indicate that proton-coupled electron transfer to the basic residue and ZnP^dot+, respectively, facilitates Y191^dot formation. Replacing Cc with the irreversible oxidant [Co(NH3)5Cl]2+ isolates distinct protein radicals for characterization by Electron Paramagnetic Resonance (EPR) spectroscopy. Radical distributions and computation indicate that W191^dot+ lies close in potential to ZnP^dot+ and that the two radicals exchange on a slow time scale despite their close separation. Remarkably, Y191:E/H232 ZnCcP variants propagate radicals differently to peripheral sites depending on the nature of the 232 residue. QM/MM calculations support radical exchange between ZnP^dot+/Trp^dot+ and the importance of a hydrogen bond to Y191^dot for maintaining a high potential to oxidize peripheral donors. These resolved reactivity patterns of CcP/ZnCcP have general relevance for engineering proton management to separate and migrate charge in proteins and potentially other molecular systems.

biochemistry↗

bioRxiv2025

An ELISA for discovering protein-protein interaction inhibitors: blocking lysinoalanine crosslinking between subunits of the spirochete flagellar hook as a test case

The inhibition of a specific protein-protein interaction is often difficult to achieve in targeted drug design. We report the development and optimization of a general-purpose, readily implemented enzyme-linked immunosorbent assay (ELISA) for high-throughput screening to identify small- molecule inhibitors of protein interactions. This ELISA does not involve the use of any capture antibodies, probes, or compounds coated on the plate and represents a general strategy to identify inhibitors of a given protein-protein interaction. We demonstrate its utility in blocking lysinoalanine crosslinking between subunits of the spirochete flagellar hook by targeting the native form of the FlgE protein, which differs from the strategies used in previous assays. The flagellar hook protein FlgE self-catalyzes the formation of a lysinoalanine (Lal) inter-subunit crosslink that is essential for the motility, and thus, infectivity of spirochetes. Prevention of Lal crosslinking through inhibition with small molecules thus represents an avenue for therapeutic development against spirochete-related diseases, such as Lyme and syphilis. Screening a library of [~]700 compounds with the ELISA confirmed that hexachlorophene, currently the only known inhibitor of Lal crosslinking in FlgE, effectively inhibits the crosslinking reaction. In addition, the assay identified two new potential inhibitors, honokiol and zafirlukast, and several activators which belong to well-known classes of antibiotics.

biochemistry↗

bioRxiv2025

Structure and dynamics of a muti-domain nitric oxide synthase regulated by a C2 domain

Nitric oxide synthase (NOS) is a widely studied multidomain redox enzyme that produces the key signaling molecule and cytotoxic agent nitric oxide (NO) for functions that range from mammalian vasodilation to prokaryotic antibiotic resistance. NOS enzymes from metazoans and cyanobacteria rely on dynamic associations of their oxygenase and coupled di-flavin reductase domains that have largely evaded detailed structural characterization. CryoEM studies of a representative dimeric six-domain Synechococcus NOS reveal the architecture of the full-length enzyme, which contains an unusual regulatory C2 domain, and additional nitric oxide deoxygenase (NOD) and pseudo-globin modules. Five distinct structural states depict how pterin binding couples to tight and loose oxygenase conformations and how the Ca2+-sensitive C2 domain moves over 85 [A] to alternatively regulate either the NOS or NOD heme center. The extended C-terminal tail and its dynamic interactions highlight an added layer of regulation required by multidomain NOSs compared to other di-flavin reductases. TeasersyNOS is a highly dynamic multi-domain oxidoreductase that harnesses a Ca2+-sensitive C2 domain to modulate activity.

biophysics↗

bioRxiv2025

Lactate dehydrogenase is the Achilles' heel of Lyme disease bacterium Borreliella burgdorferi

As a zoonotic pathogen, the Lyme disease bacterium Borreliella burgdorferi has evolved unique metabolic pathways, some of which are specific and essential for its survival and thus present as ideal targets for developing new therapeutics. B. burgdorferi dispenses with the use of thiamin as a cofactor and relies on lactate dehydrogenase (BbLDH) to convert pyruvate to lactate for balancing NADH/NAD+ ratios. This report first demonstrates that BbLDH is a canonical LDH with some unique biochemical and structural features. A loss-of-function study then reveals that BbLDH is essential for B. burgdorferi survival and infectivity, highlighting its therapeutic potential. Drug screening identifies four previously unknown LDH inhibitors with minimal cytotoxicity, two of which inhibit B. burgdorferi growth. This study provides mechanistic insights into the function of BbLDH in the pathophysiology of B. burgdorferi and lays the groundwork for developing genus-specific metabolic inhibitors against B. burgdorferi and potentially other tick-borne pathogens as well.

microbiology↗

bioRxiv2024

Flavoproteins as native and genetically encoded spin probes for in cell ESR spectroscopy

Flavin cofactors are attractive Electron Spin Resonance (ESR) probes for proteins because cellular reductants and light can generate their semiquinone states. We have used ESR spectroscopy to study the bacterial transmembrane aerotaxis receptor (Aer) in its native Escherichia coli membrane environment. Optimization of the spectroscopic (electronic relaxation times) and cell growth (isotopic labeling) conditions allowed for measurements of Aer with its partners - the histidine kinase (CheA) and the coupling protein (CheW) - in native signaling arrays. Continuous-wave ESR measurements at room temperature showed a rigid Aer flavin immobilized in the cofactor pocket and Q-band electron nuclear double resonance (ENDOR) measurements identified a predominant anionic semiquinone radical state in cell. Q-band four-pulse double electron-electron resonance (4P-DEER) measurements indicated a 4.1 nm distance between the two flavins of an Aer homodimer, consistent with previous in vitro measurements, but also revealed additional separations in cell indicative of chemoreceptor arrays, not previously observed for Aer. For general application, we further developed a genetically encoded Light-Oxygen and Voltage (LOV) domain for incorporation into target proteins as an ESR probe of structural properties in cell. This approach provides a framework to elucidate protein oligomeric states and conformations that are difficult to reproduce in vitro.

biochemistry↗

bioRxiv2022

Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock

Circadian clocks are composed of molecular oscillators that pace rhythms of gene expression to the diurnal cycle. Therein, transcriptional-translational negative feedback loops (TTFLs) generate oscillating levels of transcriptional repressor proteins that regulate their own gene expression. In the filamentous fungus Neurospora crassa, the proteins Frequency (FRQ), the FRQ-interacting RNA helicase (FRH) and Casein-Kinase I (CK1) form the FFC complex that represses expression of genes activated by the White-Collar complex (WCC). A key question concerns how FRQ orchestrates molecular interactions at the core of the clock despite containing little predicted tertiary structure. We present the reconstitution and biophysical characterization of FRQ and the FFC in unphosphorylated and highly phosphorylated states. Site-specific spin labeling and pulse- dipolar ESR spectroscopy provides domain-specific structural details on the full-length, 989- residue intrinsically disordered FRQ and the FFC. FRQ contains a compact core that associates and organizes FRH and CK1 to coordinate their roles in WCC repression. FRQ phosphorylation increases conformational flexibility and alters oligomeric state but the changes in structure and dynamics are non-uniform. Full-length FRQ undergoes liquid-liquid phase separation (LLPS) to sequester FRH and CK1 and influence CK1 enzymatic activity. Although FRQ phosphorylation favors LLPS, LLPS feeds back to reduce FRQ phosphorylation by CK1 at higher temperatures. Live imaging of Neurospora hyphae reveals FRQ foci characteristic of condensates near the nuclear periphery. Analogous clock repressor proteins in higher organisms share little position-specific sequence identity with FRQ; yet, they contain amino-acid compositions that promote LLPS. Hence, condensate formation may be a conserved feature of eukaryotic circadian clocks.

biochemistry↗