Biology subjects

Foster, M. P.

Publications and source records attributed to Foster, M. P..

11 recordsLinked to original sources

bioRxiv2026

Enhanced DNA Scanning and Target Specificity via Motif Sequestration

Sequence-specific DNA-binding proteins must efficiently locate their target sequences within large, structurally heterogeneous genomes while avoiding nonproductive off-target interactions. Here, we show that an autoinhibitory conformational state of the site-specific recombinase Cre lowers nonspecific DNA-binding affinity to promote one-dimensional DNA scanning while suppressing off-target assembly of recombinogenic complexes. Recent studies indicate that the C-terminal segment of Cre transiently adopts a sequestered conformation that occludes the catalytic domain's DNA-binding surface. Using NMR spectroscopy, single-molecule fluorescence microscopy, and optical trapping, we investigated how this conformational equilibrium regulates target search. NMR measurements reveal that the sequestered state predominates in the absence of DNA and persists at noncognate sites, whereas binding to cognate loxP sequences relieves autoinhibition. Single-molecule tracking demonstrates that full-length Cre undergoes processive one-dimensional diffusion and transitions to stable binding upon cognate-site recognition, whereas C-terminal truncation impairs scanning efficiency by arresting diffusion. These findings demonstrate how transient motif sequestration coordinates target search with site-specific complex assembly, suggesting a general mechanism by which DNA-modifying enzymes balance search efficiency and target specificity.

biophysics↗

bioRxiv2026

Thermodynamics of Indirect Readout in Cre-loxP Recognition

Cre, a site-specific tyrosine DNA recombinase, enables targeted manipulation of genetic material without production of cytotoxic double-strand DNA breaks. Though widely deployed in biotechnology, an incomplete understanding of how Cre recognizes its cognate loxP target limits its broader application in human health. Cre has been proposed to recognize an inverted pair of recombinase binding elements (RBEs) at loxP sites using an indirect readout mechanism, inducing conformations that are disfavored at noncognate sites. Despite a high degree of specificity, structural studies have shown that Cre protomers make few direct base-specific contacts to each RBE, implicating noncontacted positions in recognition by tuning flexibility and enabling binding-coupled conformational changes. We designed a set of loxP half-site variants predicted to rigidify the DNA substrate and measured the thermodynamics of Cre binding by isothermal titration calorimetry. Thermodynamic signatures and NMR spectra reveal that unfavorable mutations at noncontacted positions in the RBE reduce binding- coupled conformational changes. Moreover, mass photometry of Cre binding to oligonucleotides containing two properly spaced RBEs revealed that non-contacted positions in the intervening 8-base pair spacer influence cooperative dimerization of two Cre protomers at loxP sites, and their synapsis to form catalytically active Cre4-loxP2 complexes. These results demonstrate that noncontacted positions contribute to specificity by encoding favorable DNA mechanics, offering new design principles for engineering Cre variants that target alternative DNA sequences.

biophysics↗

bioRxiv2025

Simultaneous ligand binding to intact and partially formed ATP binding sites in the hexameric termination factor Rho

Thermodynamic coupling between ligand binding sites affords macromolecular machines a means to coordinate processive function. Because these machines may be compositionally complex, quantifying and interpreting ligand binding events can be experimentally difficult. Biophysical methods that convolve binding events into a one-dimensional metric, which suffice for monomeric macromolecules that bind to a single ligand, are insufficient to adequately describe the complexity of binding to oligomeric systems. Confounding factors include structural heterogeneity that may invalidate basic assumptions used to interpret the measurements. In this communication, we use native mass spectrometry to measure ATP binding to a hexameric helicase, the E. coli termination factor Rho. Providing new insights into classical and more recent biochemical experiments, we observe and quantify ATP binding to hexameric and lower-order complexes. Moreover, we observe super-stoichiometric binding consistent with ATP binding to partially formed binding sites at the edges of the open washer structure. Such detailed insights are likely critical to understanding the mechanisms by which a broad range of macromolecular machines harness the free energy from ligand binding, hydrolysis, and exchange to coordinate their ligand-dependent functions.

biophysics↗

bioRxiv2024

Protein and DNA Conformational Changes Contribute to Specificity of Cre Recombinase

Cre, a conservative site-specific tyrosine recombinase, is a powerful gene editing tool in the laboratory. Expanded applications in human health are hindered by lack of understanding of the mechanism by which Cre selectively binds and recombines its cognate loxP sequences. This knowledge is essential for retargeting the enzyme to new sites and for mitigating effects of off-target recombination. Prior studies have suggested that in addition to a few base-specific contacts to cognate loxP DNA, the enzymes specificity is enhanced by (1) autoinhibition involving a conformational change in the proteins C-terminal helix, and (2) indirect readout via binding-coupled conformational changes in the target DNA. We used isothermal titration calorimetry (ITC), circular dichroism (CD) and heteronuclear NMR spectroscopy to investigate DNA site recognition by wild-type Cre and a deletion mutant lacking the C-terminal helix. ITC of Cre and a C-terminal deletion variant against cognate and non-cognate DNA recombinase binding elements (RBEs) reveal that the C-terminus reduces DNA binding affinity by six-fold towards cognate DNA. Additionally, ITC revealed highly unfavorable binding enthalpy, which when combined with evidence from CD and NMR of structural differences between cognate and non-cognate complexes support a model in which binding-coupled DNA bending provides a unique structure-thermodynamic signature of cognate complexes. Together, these findings advance our understanding of site-recognition by Cre recombinase. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/627928v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@19fb34dorg.highwire.dtl.DTLVardef@1b4b025org.highwire.dtl.DTLVardef@1988254org.highwire.dtl.DTLVardef@1945cb1_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Conformational dynamics in specialized C2H2 zinc finger domains enable zinc-responsive gene repression in S. pombe

Loz1 is a zinc-responsive transcription factor in fission yeast that maintains cellular zinc homeostasis by repressing the expression of genes required for zinc uptake in high zinc conditions. Previous deletion analysis of Loz1 found a region containing two tandem C2H2 zinc-fingers and an upstream "accessory domain" rich in histidine, lysine, and arginine residues to be sufficient for zinc-dependent DNA binding and gene repression. Here we report unexpected biophysical properties of this pair of seemingly classical C2H2 zinc fingers. Isothermal titration calorimetry and NMR spectroscopy reveal two distinct zinc binding events localized to the zinc fingers. NMR spectra reveal complex dynamic behavior in this zinc responsive region spanning time scales from fast 10-12-10-10 to slow > 100 sec. Slow exchange due to cis-trans isomerization of the TGERP linker results in doubling of many signals in the protein. Conformational exchange on the 10-3 s timescale throughout the first zinc finger distinguishes it from the second and is linked to weaker affinity for zinc. These findings reveal the mechanism of zinc sensing by Loz1 and illuminate how the proteins rough free-energy landscape enable zinc sensing, DNA binding and regulated gene expression. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/614115v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@8bb7ddorg.highwire.dtl.DTLVardef@d43153org.highwire.dtl.DTLVardef@f25930org.highwire.dtl.DTLVardef@bd7c8b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Transmembrane channel-like 4 and 5 proteins at microvillar tips are potential ion channels and lipid scramblases

Microvilli--membrane bound actin protrusions on the surface of epithelial cells--are sites of critical processes including absorption, secretion, and adhesion. Increasing evidence suggests microvilli are mechanosensitive, but underlying molecules and mechanisms remain unknown. Here, we localize transmembrane channel-like proteins 4 and 5 (TMC4 and 5) and calcium and integrin binding protein 3 (CIB3) to microvillar tips in intestinal epithelial cells, near glycocalyx insertion sites. We find that TMC5 colocalizes with CIB3 in cultured cells and that a TMC5 fragment forms a complex with CIB3 in vitro. Homology and AlphaFold2 models reveal a putative ion permeation pathway in TMC4 and 5, and molecular dynamics simulations predict both proteins can conduct ions and perform lipid scrambling. These findings raise the possibility that TMC4 and 5 interact with CIB3 at microvillar tips to form a mechanosensitive complex, akin to TMC1 and 2, and CIB2 and 3, within the mechanotransduction channel complex at the tips of inner ear stereocilia.

cell biology↗

bioRxiv2024

Insights into Ligand-Mediated Activation of an Oligomeric Ring-Shaped Gene-Regulatory Protein from Solution- and Solid-State NMR

The 91 kDa oligomeric ring-shaped ligand binding protein TRAP (trp RNA binding attenuation protein) regulates the expression of a series of genes involved in tryptophan (Trp) biosynthesis in bacilli. When cellular Trp levels rise, the free amino acid binds to sites buried in the interfaces between each of the 11 (or 12, depending on the species) protomers in the ring. Crystal structures of Trp-bound TRAP show the Trp ligands are sequestered from solvent by a pair of loops from adjacent protomers that bury the bound ligand via polar contacts to several threonine residues. Binding of the Trp ligands occurs cooperatively, such that successive binding events occur with higher apparent affinity but the structural basis for this cooperativity is poorly understood. We used solution methyl-TROSY NMR relaxation experiments focused on threonine and isoleucine sidechains, as well as magic angle spinning solid-state NMR 13C-13C and 15N-13C chemical shift correlation spectra on uniformly labeled samples recorded at 800 and 1200 MHz, to characterize the structure and dynamics of the protein. Methyl 13C relaxation dispersion experiments on ligand-free apo TRAP revealed concerted exchange dynamics on the {micro}s-ms time scale, consistent with transient sampling of conformations that could allow ligand binding. Cross-correlated relaxation experiments revealed widespread disorder on fast timescales. Chemical shifts for methyl-bearing side chains in apo- and Trp-bound TRAP revealed subtle changes in the distribution of sampled sidechain rotameric states. These observations reveal a pathway and mechanism for induced conformational changes to generate homotropic Trp-Trp binding cooperativity.

biophysics↗

bioRxiv2024

Structural basis of nearest-neighbor cooperativity in the ring-shaped gene regulatory protein TRAP from protein engineering and cryo-EM

Homotropic cooperativity is widespread in biological regulation. The homo-oligomeric ring-shaped trp RNA binding attenuation protein (TRAP) from bacillus binds multiple tryptophan ligands (Trp) and becomes activated to bind a specific sequence in the 5 leader region of the trp operon mRNA. Ligand-activated binding to this specific RNA sequence regulates downstream biosynthesis of Trp in a feedback loop. Characterized TRAP variants form 11- or 12-mer rings and bind Trp at the interface between adjacent subunits. Various studies have shown that a pair of loops that gate each Trp binding site is flexible in the absence of the ligand and become ordered upon ligand binding. Thermodynamic measurements of Trp binding have revealed a range of cooperative behavior for different TRAP variants, even if the averaged apparent affinities for Trp have been found to be similar. Proximity between the ligand binding sites, and the ligand-coupled disorder-to-order transition has implicated nearest-neighbor interactions in cooperativity. To establish a solid basis for describing nearest-neighbor cooperativity we engineered dodecameric (12-mer) TRAP variants constructed with two subunits connected by a flexible linker (dTRAP). We mutated one of the protomers such that only every other site was competent for Trp binding. Thermodynamic and structural studies using native mass spectrometry, NMR spectroscopy, and cryo-EM provided unprecedented detail into the thermodynamic and structural basis for the observed ligand binding cooperativity. Such insights can be useful for understanding allosteric control networks and for the development of new ones with defined ligand sensitivity and regulatory control. SignificanceHomo-oligomeric proteins are ubiquitous in biology, and their function is often regulated by activator ligands that bind cooperatively, via poorly understood mechanisms. TRAP is a bacterial protein responsible for sensing changes in cellular tryptophan concentration, and for downregulating its production when levels are elevated. By fusing two subunits via a flexible linker, we assembled protein rings in which every other of its twelve ligand binding sites is incapable of binding. We used calorimetry and native mass spectrometry to measure ligand binding and found that these nearest-neighbor interactions are essential for cooperative ligand binding. Cryo-EM of these engineered proteins provided the means to observe the structure-thermodynamic linkage essential for understanding ligand binding cooperativity.

biophysics↗

bioRxiv2023

Complexes of vertebrate TMC1/2 and CIB2/3 proteins form hair-cell mechanotransduction cation channels

Calcium and integrin-binding protein 2 (CIB2) and CIB3 bind to transmembrane channel-like 1 (TMC1) and TMC2, the pore-forming subunits of the inner-ear mechano-electrical transduction (MET) apparatus. These interactions have been proposed to be functionally relevant across mechanosensory organs and vertebrate species. Here we show that both CIB2 and CIB3 can form heteromeric complexes with TMC1 and TMC2 and are integral for MET function in mouse cochlea and vestibular end organs as well as in zebrafish inner ear and lateral line. Our AlphaFold 2 models suggest that vertebrate CIB proteins can simultaneously interact with at least two cytoplasmic domains of TMC1 and TMC2 as validated using nuclear magnetic resonance spectroscopy of TMC1 fragments interacting with CIB2 and CIB3. Molecular dynamics simulations of TMC1/2 complexes with CIB2/3 predict that TMCs are structurally stabilized by CIB proteins to form cation channels. Overall, our work demonstrates that intact CIB2/3 and TMC1/2 complexes are integral to hair-cell MET function in vertebrate mechanosensory epithelia.

neuroscience↗

bioRxiv2022

Mechanism of homotropic cooperativity from nearest-neighbor statistical thermodynamic modelling and native mass spectrometry of ring-shaped homo-oligomeric ligand binding proteins

Homo-oligomeric ligand-activated proteins are ubiquitous in biology. The functions of such molecules are commonly regulated by allosteric coupling between ligand binding sites. Understanding the basis for this regulation requires both quantifying the free energy {Delta}G transduced between sites, and the structural basis by which it is transduced. We consider allostery in three variants of the model ring-shaped homo-oligomeric trp RNA binding attenuation protein, TRAP. First, we developed nearest-neighbor statistical thermodynamic binding models comprising microscopic free energies for ligand binding to isolated sites {Delta}GN0, and for coupling between one or both adjacent sites, {Delta}GN1 and {Delta}GN2. Using the resulting partition function (PF) we explored the effects of these parameters on simulated population distributions for the 2N possible liganded states. We then experimentally monitored liganddependent population shifts using conventional spectroscopic and calorimetric methods, and using native mass spectrometry (MS). By resolving species with differing numbers of bound ligands by their mass, native MS revealed striking differences in their ligand-dependent population shifts. Fitting the populations to a binding polynomial derived from the PF yielded coupling free energy terms corresponding to orders of magnitude differences in cooperativity. Uniquely, this approach predicts which of the possible 2N liganded states are populated at different ligand concentrations, providing necessary insights into regulation. The combination of statistical thermodynamic modeling with native MS may provide the thermodynamic foundation for a meaningful understanding of the structure-thermodynamic linkage that drives cooperativity. TOC Figure (draft) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/484990v3_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@2ce1f3org.highwire.dtl.DTLVardef@a6541borg.highwire.dtl.DTLVardef@1dbd08aorg.highwire.dtl.DTLVardef@58297e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOTOC Figure.C_FLOATNO Ligand (Trp) binding to multiple sites on homo-oligomeric ring-shaped proteins like TRAP alters their functional states. Homotropic cooperativity is expected to alter the activation pathway in response to cellular ligand concentration. In the presence of positive nearest-neighbor cooperativity, ligand binding is favored at adjacent sites, whereas in the absence of cooperativity, a random "Normal" distribution is expected. C_FIG

biophysics↗

bioRxiv2021

Nearest-neighbor effects modulate loxP spacer DNA chemical shifts and guide oligonucleotide design for NMR studies

Cre recombinase catalyzes site-specific DNA recombination at pseudo-palindromic loxP sites through two rounds of strand cleavage, exchange, and religation. Cre is a potential gene editing tool of interest due its lack of requirements for external energy sources or host factors, as well as the fact that it does not generate potentially cytotoxic double-stranded DNA breaks. However, broader applications of Cre in editing noncanonical target sequences requires a deeper understanding of the DNA features that enable target site selection and efficient recombination. Although Cre recombines loxP DNA in a specific and ordered fashion, it makes few direct contacts to the loxP spacer, the region where recombination occurs. Furthermore, little is known about the structural and dynamic features of the loxP spacer that make it a suitable target for Cre. To enable NMR spectroscopic studies of the spacer, we have aimed to identify a fragment of the 34-bp loxP site that retains the structural features of the spacer while minimizing the spectral crowding and line-broadening seen in longer oligonucleotides. We report sequential backbone resonance assignments for loxP oligonucleotides of varying lengths and evaluate chemical shift differences, {Delta}{delta}, between the oligos. Analysis of flanking sequence effects and mutations on spacer chemical shifts indicates that nearest-neighbor and next-nearest-neighbor effects dominate the chemical environment experienced by the spacer. We have identified a 16-bp oligonucleotide that adequately preserves the structural environment of the spacer, setting the stage for NMR-based structure determination and dynamics investigations.

biochemistry↗