Biology subjects

Stachowski, K.

Publications and source records attributed to Stachowski, K..

5 recordsLinked to original sources

bioRxiv2025

End-to-end automation of repeat-target cryo-EM structure determination in CryoSPARC

Single particle cryo-EM is a valuable and growing technique for life science and drug discovery. Currently, obtaining state-of-the-art results from cryo-EM data analysis requires a human in the loop to analyze intermediate results and make image processing decisions. This bottleneck limits the achievable throughput of structure determination, especially in high-throughput settings such as structure-based drug design. In this work, we develop an end-to-end automation strategy for repeat-target structure determination using new tools in CryoSPARC. We demonstrate completely hands-off processing of 21 challenging G protein-coupled receptor (GPCR) datasets. In 17 of 21 cases, automated processing meets or exceeds published resolution and map quality and, in several cases, provides significant improvement in receptor and ligand density that allows improved model building. Our results on both active and inactive state GPCRs show that our automation strategy generalizes easily to new target classes, and that complete automation of data processing is straightforward to achieve in CryoSPARC. We provide downloadable CryoSPARC Workflow files so that users can import, replicate, adapt and extend our automated workflow for their own targets, enabling cryo-EM to be applied at larger scales and to answer larger biological questions.

biophysics↗

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

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↗

bioRxiv2021

Unloxing the assembly and activation mechanism of Cre recombinase using Cryo-EM

Mechanistic understanding of the structural basis for DNA recombination in the Cre-loxP system has largely been guided by crystallographic structures of tetrameric synaptic complexes (intasomes). These structural and biochemical studies have suggested that conformational changes and DNA bending in presynaptic complexes underlie site-selection and activation mechanisms of Cre recombinase. Here we used protein engineering and various DNA substrates to isolate the Cre-loxP (54 kDa), Cre2-loxP (110 kDa), and Cre4-loxP2 assembly intermediates, and determined their structures using cryo-EM to resolutions of 3.9 [A], 4.5 [A], and 3.2 [A], respectively. Progressive DNA bending along the assembly pathway enables formation of increasingly intimate protein-protein interfaces. Insufficient stabilization of important protein motifs observed during the assembly process provides a compelling explanation for the observed half-the-sites activity, and preferential bottom strand cleavage of loxP sequences. We found that selection of loxP sites is largely dependent on Cres ability to bend and stabilize the spacer region between two recombinase binding elements. Application of 3D variability analysis to the tetramer data reveals a propensity for motion along the pathway between protomer activation and Holliday junction isomerization. These findings help us to better understand loxP site specificity, controlled activation of alternating protomers, the basis for the observed bias of strand cleavage order, and the importance of conformational sampling, especially with regards to site-selection and activity among Cre variants. Furthermore, our findings provide invaluable information for the rational development of designer, site-specific recombinases for use as gene editing technologies. HighlightsO_LICryo-EM structures of Cre-loxP assembly intermediates (monomer, dimer, and tetramer) provide insights into mechanisms of site recognition, half-the-sites activity, strand cleavage order, and concerted strand cleavage C_LIO_LISelectivity of loxP sites arises from few base-specific contacts made by each protomer and is mainly driven by formation of phosphate contacts and DNA deformations that are maximal in the fully assembled "active" tetramer C_LIO_LICis and trans interactions of the {beta}2-3 loop (i) define which sites are "active" and (ii) ensure half-the-sites activity C_LIO_LIProtein flexibility plays a central role in enabling DNA sequence scanning, recruitment of a second protein to form a dimer, synapsis, control of activity, as well as subsequent recombination steps C_LIO_LIConformational sampling within the tetrameric complex was uncovered using 3D variability analysis and revealed the importance of protein-protein interfaces for site- selection and activation of Cre-loxP complexes C_LI

biochemistry↗

bioRxiv2020

DNA Binding Induces a cis to trans Switch in Cre Recombinase to Enable Intasome Assembly

Mechanistic understanding of DNA recombination in the Cre-loxP system has largely been guided by crystallographic structures of tetrameric synaptic complexes. Those studies have suggested a role for protein conformational dynamics that has not been well characterized at the atomic level. We used solution NMR to discover the link between intrinsic flexibility and function in Cre recombinase. TROSY-NMR spectra show the N-terminal and C-terminal catalytic domains (CreNTD, CreCat) to be structurally independent. Amide 15N relaxation measurements of the CreCat domain reveal fast time scale dynamics in most regions that exhibit conformational differences in active and inactive Cre protomers in crystallographic tetramers. However, the C-terminal helix N, implicated in assembly of synaptic complexes and regulation of DNA cleavage activity via trans protein-protein interactions, is unexpectedly rigid in free Cre. Chemical shift perturbations and intra- and inter-molecular paramagnetic relaxation enhancement (PRE) NMR data reveal an alternative auto-inhibitory conformation for the N region of free Cre, wherein it packs in cis over the protein DNA binding surface and active site. Moreover, binding to loxP DNA induces a conformational change that dislodge the C-terminus, resulting in a cis to trans switch that is likely to enable protein-protein interactions required for assembly of recombinogenic Cre intasomes. These findings necessitate a re-examination of the mechanisms by which this widely-utilized gene editing tool selects target sites, avoids spurious DNA cleavage activity, and controls DNA recombination efficiency. SIGNIFICANCE STATEMENTThe Cre-loxP system is a widely used gene editing tool that has enabled transformative advances in immunology, neuroscience and cardiovascular research. Still, off-target activities confound research results and present obstacles to biomedical applications. Overcoming those limitations requires understanding the steps leading to assembly of recombination complexes, intasomes. We measured the magnetic properties of nitrogen nuclei in the backbone of the enzyme to correlate its intrinsic dynamics with its function in DNA recognition and cleavage. Remarkably, we found that in the absence of DNA the C-terminus of Cre appears to block the DNA binding surface and active site of the enzyme. Binding to loxP DNA induces a conformational switch that would enable the intermolecular protein-protein interactions required for assembly of recombinogenic Cre intasomes.

biophysics↗