Biology subjects

Kabasakal, B. V.

Publications and source records attributed to Kabasakal, B. V..

9 recordsLinked to original sources

bioRxiv2026

Structural heterogeneity of uncrosslinked FtsH-HflKC assemblies includes variable FtsH occupancy and a distinct wild-type FtsH architecture

The AAA+ metalloprotease FtsH associates with HflK and HflC to form the megadalton FtsH-HflKC complex (FHC), which contributes to membrane-protein quality control in Escherichia coli. Recent structural studies have revealed asymmetric HflKC architectures and variable FtsH occupancy, contrasting with earlier crosslinked structures of a symmetric four-hexamer cage. Here, we analyzed wild-type, uncrosslinked FHC co-expressed from a single multicistronic plasmid and solubilized in lauryl maltose neopentyl glycol (LMNG) using single-particle cryo-EM, SEC-SAXS, and biochemical activity assays. Two-dimensional classification identified HflKC-containing particles associated with one or two FtsH hexamers, together with a distinct population of isolated FtsH particles. Focused reconstruction of the FtsH cytosolic domain reached 5.32 angstrom resolution and provided a structural view of wild-type, uncrosslinked FtsH without stabilizing substitutions or an added nucleotide analogue. The reconstruction retained the characteristic stacked arrangement of the AAA+ and protease rings but showed a more laterally splayed AAA+ ring compared with the stabilized reference, while the protease ring remained broadly similar in overall envelope. A 9.63 angstrom HflKC consensus reconstruction and 3D variability analysis (3DVA) in C1, further revealed substantial conformational variability spanning open-like and more constricted scaffold geometries. SEC-SAXS of a selected later-eluting LMNG-solubilized fraction yielded a compact anisotropic envelope with an estimated Dmax of ~110-115 angstrom, although this species could not be assigned to a defined intact FHC stoichiometry. Under the conditions tested, the FHC preparation also showed lower apparent ATPase and proteolytic activities than isolated FtsH. Together, these findings highlight the compositional and conformational heterogeneity of uncrosslinked FHC and reveal a more laterally splayed AAA+ arrangement in wild-type FtsH compared with the stabilized reference structure.

biophysics↗

bioRxiv2025

Cryo-EM structure of the FtsH periplasmic domain reveals functional dynamics

FtsH, an essential AAA+ metalloprotease, maintains cellular homeostasis by degrading misfolded and membrane-associated proteins. Here, we report cryo-EM structures of the Escherichia coli FtsH periplasmic domain (FtsH-PD) revealing insights into its conformational flexibility. Initial 2D class averages suggested three distinct orientations, right-handed and left-handed maps of FtsH-PD, and a map with a different conformation. The 4.9 [A] structure of FtsH-PD exhibits the conserved +{beta} fold, while the 7.3 [A] map with the different conformation displays a 20{o} clockwise rotation of two alpha helices. These findings support a model where conformational changes are present not only in the FtsH cytosolic domain, but also in the periplasmic domain and potentially facilitate substrate translocation through a combination of mechanisms involving both the FtsH-PD and the HflKC complexed with FtsH, along with lipid-scramblase activity to assist in membrane protein extraction. This study points out novel perspectives on how conformational changes in the periplasmic domain contribute to FtsH substrate degradation mechanisms.

biochemistry↗

bioRxiv2025

Off-Target Structural Insights: ArnA and AcrB in Bacterial Membrane Protein Cryo-EM Analysis

Membrane protein quality control in Escherichia coli involves coordinated actions of the AAA+ protease FtsH, the insertase YidC, and the regulatory complex HflKC. These systems maintain proteostasis by facilitating membrane protein insertion, folding, and degradation. To gain structural insights into a putative complex formed by FtsH and YidC, we performed single-particle cryogenic electron microscopy on detergent-solubilized membrane samples, from which FtsH and YidC were purified using Ni-NTA affinity and size exclusion chromatography. Although SDS-PAGE analysis indicated a high purity of these proteins, cryo-EM datasets unexpectedly yielded high-resolution structures of ArnA and AcrB at 4.0 [A] and 2.9 [A] resolutions, respectivelyArnA is a bifunctional enzyme involved in lipid A modification and polymyxin resistance, while AcrB is a multidrug efflux transporter of the AcrAB-TolC system. ArnA and AcrB are known membrane protein contaminants after Ni-NTA purifications. However, they were also detected in Strep-Tactin affinity purified FtsH-YidC samples. ArnA, typically cytoplasmic, was consistently found in membrane-isolated samples, indicating an association with membrane components. Two-dimensional class averages revealed additional particles resembling GroEL, cytochrome bo oxidase, and the AAA+ cytoplasmic domain of FtsH. Although our preparation was initially considered homogeneous, neither full-size FtsH nor the intact FtsH-YidC complex could be resolved. This case highlights that cryo-EM can reveal structural information beyond the intended target, and that overexpression of membrane proteins involved in quality control may lead to the co-purification of proteins such as ArnA and AcrB.

biochemistry↗

bioRxiv2024

The crystal structure of the Shetha Protein II (FeSII) from Azotobacter vinelandii suggests a domain swap

The Azotobacter vinelandii FeSII protein forms an oxygen resistant complex with the nitrogenase MoFe and Fe proteins. FeSII is an adrenodoxin type ferredoxin, forming a dimer in solution. Previously, the crystal structure was solved (Schlesier et al., 2016), with 5 copies in the asymmetric unit. One copy is a normal adrenodoxin domain, forming a dimer with its crystallographic symmetry mate. The other four copies are in an "open" conformation with a loop flipped out exposing the 2Fe-2S cluster. The open and closed conformations were interpreted as oxidised and reduced, and the large conformational change in the open configuration allowed binding to nitrogenase. We have independently solved the structure of FeSII in the same crystal form. Our positioning of the atoms in the unit cell is similar to the earlier report. However, our interpretation of the structure is different. We interpret the "open" conformation as the product of a crystallization-induced domain swap. The 2Fe-2S cluster is not exposed to solvent, but in the crystal its interacting helix is replaced by the same helix residues from a crystal symmetry mate. The domain swap is complicated, as it is unusual in being in the middle of the protein rather than at a terminus, and it creates arrangements of molecules that can be interpreted in multiple ways. We caution that crystal structures should be interpreted in terms of the contents of the entire crystal rather than one asymmetric unit.

biochemistry↗

bioRxiv2023

Combined workflow for XL-MS data processing: Application to the FtsH-HflK-HflC membrane protein complex

Recent advances in mass spectrometry (MS) yielding sensitive and accurate measurements along with developments in software tools have enabled the characterization of complex systems routinely. Thus, structural proteomics and cross-linking mass spectrometry (XL-MS) have become a useful method for structural modeling of protein complexes. Here we utilized commonly used XL-MS software tools to elucidate the protein interactions within a membrane protein complex containing FtsH, HflK, and HflC, over-expressed in E.coli. The MS data were processed using MaxLynx, MeroX, MS Annika, xiSEARCH, and XlinkX software tools. The number of identified inter- and intra-protein cross-links varied among software. Each interaction was manually checked using the raw MS and MS/MS data and distance restraints to verify inter- and intra-protein cross-links. A total of 37 inter-protein and 148 intra-protein cross-links were determined in the FtsH-HflK-HflC complex. The 59 of them were new interactions on the lacking region of recently published structures. These newly identified interactions, when combined with molecular docking and structural modeling, present opportunities for further investigation. The results provide valuable information regarding the complex structure and function to decipher the intricate molecular mechanisms underlying the mega FtsH-HflK-HflC complex. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/544024v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@8e10f5org.highwire.dtl.DTLVardef@fb809borg.highwire.dtl.DTLVardef@1b84df3org.highwire.dtl.DTLVardef@105c8e6_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO The overall workflow of XL-MS analysis: the steps of XL-MS studies applied to the two samples taken from different stages of purification process of mega membrane protein complex FtsH-HflC-HflK. C_FIG

biochemistry↗

bioRxiv2023

Dynamic lid domain of Chloroflexus aurantiacus Malonyl-CoA Reductase controls the reaction

Malonyl-Coenzyme A Reductase (MCR) in Chloroflexus aurantiacus, a characteristic enzyme of the 3-hydroxypropionate (3-HP) cycle, catalyses the reduction of malonyl-CoA to 3-HP. MCR is a bi-functional enzyme; in the first step, malonyl-CoA is reduced to the free intermediate malonate semialdehyde by the C-terminal region of MCR, and further reduced to 3-HP by the N-terminal region of MCR. Here we present the crystal structures of both N-terminal and C-terminal regions of the split MCR from C. aurantiacus. A catalytic mechanism is suggested by ligand and substrate bound structures, and structural and kinetic studies of MCR variants. Both MCR structures reveal one catalytic, and one non-catalytic SDR (short chain dehydrogenase/reductase) domain. C-terminal MCR has a lid domain which undergoes a conformational change and controls the reaction. In the proposed mechanism of the C-terminal MCR, the conversion of malonyl-CoA to malonate semialdehyde is based on the reduction of malonyl-CoA by NADPH, followed by the decomposition of the hemithioacetal to produce malonate semialdehyde and coenzyme A. Conserved arginines, Arg734 and Arg773 are proposed to play key roles in the mechanism and conserved Ser719, and Tyr737 are other essential residues forming an oxyanion hole for the substrate intermediates.

biochemistry↗

bioRxiv2022

Rapid and High Resolution Ambient Temperature Structure Determination at Turkish Light Source

High-resolution biomacromolecular structure determination is essential to better understand protein function and dynamics. Serial crystallography is an emerging structural biology technique which has fundamental limitations due to either sample volume requirements or immediate access to the competitive X-ray beamtime. Obtaining a high volume of well-diffracting, sufficient-size crystals while mitigating radiation damage remains a critical bottleneck of serial crystallography. As an alternative, we introduce the plate-reader module adapted for using a 72-well Terasaki plate for biomacromolecule structure determination at a convenience of a home X-ray source. We also present the first ambient temperature lysozyme structure determined at the Turkish Light Source (Turkish DeLight). The complete dataset was collected in 18.5 mins with resolution extending to 2.39 [A] and 100% completeness. Combined with our previous cryogenic structure (PDB ID: 7Y6A), the ambient temperature structure provides invaluable information about the structural dynamics of the lysozyme. Turkish DeLight provides robust and rapid ambient temperature biomacromolecular structure determination with limited radiation damage.

biophysics↗

bioRxiv2022

Cryogenic X-ray crystallographic studies of biomacromolecules at Turkish Light Source: Turkish DeLight

X-ray crystallography is a robust and powerful structural biology technique that provides high-resolution atomic structures of biomacromolecules. Scientists use this technique to unravel mechanistic and structural details of biological macromolecules (e.g. proteins, nucleic acids, protein complexes, protein-nucleic acid complexes, or large biological compartments). Since its inception, single-crystal cryo-crystallography has never been performed in Turkiye due to the lack of a single-crystal X-ray diffractometer. The X-ray diffraction facility recently established at the University of Health Sciences, Istanbul, Turkiye will enable Turkish and international researchers to easily perform high-resolution structural analysis of biomacromolecules from single crystals. Here, we describe the technical and practical outlook of a state-of-the-art home-source X-ray, using lysozyme as a model protein. The methods and practice described in this article can be applied to any biological sample for structural studies. Therefore, this article will be a valuable practical guide from sample preparation to data analysis.

biophysics↗

bioRxiv2018

Structural basis of light-induced redox regulation in the Calvin cycle

In plants, carbon dioxide is fixed via the Calvin cycle in a tightly regulated process. Key to this regulation is the conditionally disordered protein CP12. CP12 forms a complex with two Calvin cycle enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), inhibiting their activities. The mode of CP12 action was unknown. By solving crystal structures of CP12 bound to GAPDH, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide pre-orders CP12 prior to binding the PRK active site. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our model explains how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation.\n\nOne Sentence SummaryHow plants turn off carbon fixation in the dark.

biochemistry↗