Biology subjects

Borcik, C. G.

Publications and source records attributed to Borcik, C. G..

6 recordsLinked to original sources

bioRxiv2026

Protonated Structure of EmrE Reveals C-terminal Tail Gating Mechanism

The multidrug efflux pump EmrE is one of the smallest known active transporters and has become a model system for studying multidrug recognition and transport. While recent high-resolution structures have illuminated its dynamic substrate binding pocket, the conformations of its interhelical loops and C-terminal tail, regions critical for controlling proton coupling and gating, remain poorly characterized. Here, we report the high-resolution structure of protonated S64V EmrE determined using solution and solid-state NMR data. This new structural model shows the C-terminal tail occluding the open face of the transport pore, providing a structural basis for how EmrE minimizes proton leak in the absence of substrate. These findings support growing evidence that relatively simple model transporters must leverage an occluded state during alternating access to avoid physiologically unfavorable proton leak.

biophysics↗

bioRxiv2026

In vitro-prepared A30P alpha-synuclein fibrils adopt the conserved and disease-relevant Greek key fold

The pathological hallmark of Parkinson Disease (PD) is the formation of the protein alpha-synuclein (Asyn) into {beta}-sheet rich, self-templating fibrils in the brain. Since the first atomic structure of wild-type Asyn fibrils was determined nearly a decade ago, several other in vitro structures of hereditary mutant fibrils and structures derived from post-mortem diseased patient tissue have been determined by solid-state nuclear magnetic resonance (SSNMR) spectroscopy and cryo-electron microscopy. These structures have not only expanded the library of structures available for computational modeling of drug binding and therapeutics development but have also given unprecedented insight into the disease specificity and structural polymorphism of Asyn fibrils. Here, we report the high-resolution SSNMR structure of the A30P hereditary mutant Asyn fibril, associated with early-onset PD. Our structural model is calculated using several thousand distance restraints derived from one sample, primarily sourced through 3D 13C-13C-13C correlation experiments. The structure adopts a Greek key topology yet does not include the P30 mutation site within the fibril core. We also introduce a comprehensive method for the rapid comparison of SSNMR spectra between Asyn polymorphs of known structure and validate the A30P fold. Lastly, we find that the structure is highly similar to many other experimental structures of both in vitro and ex vivo Asyn fibrils, including those with other hereditary point mutations, suggesting a conserved accessible fold.

biophysics↗

bioRxiv2025

Alpha-Synuclein Fibril Structures Cluster into Distinct Classes

The accumulation of Alpha-synuclein (Asyn) fibrils is the defining pathologic feature in Parkinson Disease (PD), Lewy Body Dementia (LBD), and Multiple System Atrophy (MSA). As such, the process of Asyn fibril formation has been an important research area and fibrils themselves have become attractive targets for disease diagnosis and therapeutic intervention. Due to the presence of mixed populations of fibrillar proteins associated with neurodegenerative diseases in brain tissue, high-resolution structures of Asyn fibrils are essential for the design of high-specificity imaging and therapeutic agents. Approximately one hundred high-resolution solid-state NMR (SSNMR) spectroscopy and cryo-electron microscopy (cryo-EM) structures of Asyn fibrils have been deposited to the Protein Databank (PDB); intriguingly there is significant polymorphism among them. Understanding the molecular makeup and characteristic features of each structural polymorph can determine conserved structural motifs which can be used as templates to design ligands with high specificity for clinical use. Utilizing standard alignment tools and density-based clustering approaches, we objectively classify fibril structures by tertiary structure type. We find that 81% of the structures cluster into two polymorph classes. Within each class, additional subtle variations are observed which position sidechains in specific, conserved orientations, well poised as druggable targets. Furthermore, we find that the conserved structural motifs associated with each class are found in all but one published Asyn fibril structure. We consider these classifications and conserved motifs in the context of disease-relevant fibril structures and offer a perspective on the utility of in vitro fibrils as substrates for drug development and models for disease pathogenesis.

biophysics↗

bioRxiv2024

High-Resolution Cryo-EM Structure Determination of α-Synuclein - A Prototypical Amyloid Fibril

The physiological role of -synuclein (-syn), an intrinsically disordered presynaptic neuronal protein, is believed to impact the release of neurotransmitters through interactions with the SNARE complex. However, under certain cellular conditions that are not well understood, -syn will self-assemble into {beta}-sheet rich fibrils that accumulate and form insoluble neuronal inclusions. Studies of patient derived brain tissues have concluded that these inclusions are associated with Parkinsons disease, the second most common neurodegenerative disorder, and other synuclein related diseases called synucleinopathies. In addition, repetitions of and specific mutations to the SNCA gene, the gene that encodes -syn, results in an increased disposition for synucleinopathies. The latest advances in cryo-EM structure determination and real-space helical reconstruction methods have resulted in over 60 in vitro structures of -syn fibrils solved to date, with a handful of these reaching a resolution below 2.5 [A]. Here, we provide a protocol for -syn protein expression, purification, and fibrilization. We detail how sample quality is assessed by negative stain transmission electron microscopy (NS-TEM) analysis and followed by sample vitrification using the Vitrobot Mark IV vitrification robot. We provide a detailed step by step protocol for high resolution cryo-EM structure determination of -syn fibrils using RELION and a series of specialized helical reconstruction tools that can be run within RELION. Finally, we detail how ChimeraX, Coot, and Phenix are used to build and refine a molecular model into the high resolution cryo-EM map. This workflow resulted in a 2.04 [A] structure of -syn fibrils with excellent resolution of residues 36 to 97 and an additional island of density for residues 15 to 22 that had not been previously reported. This workflow should serve as a starting point for individuals new to the neurodegeneration and structural biology fields. Together, this procedure lays the foundation for advanced structural studies of -synuclein and other amyloid fibrils. Key FeaturesO_LIIn vitro fibril amplification method yielding twisting fibrils that span several micrometers in length and are suitable for cryo-EM structure determination. C_LIO_LIHigh-throughput cryo-EM data collection of neurodegenerative fibrils, such as alpha-synuclein. C_LIO_LIUse of RELION implementations of helical reconstruction algorithms to generate high-resolution 3D structures of a-synuclein fibrils. C_LIO_LIBrief demonstration of the use of ChimeraX, Coot, and Phenix for molecular model building and refinement.s C_LI Graphical overview of -synuclein fibrilization and cryo-EM structure determination-synuclein protein expression and purification is followed by a fibrilization protocol yielding twisting filaments that span several micrometers in length and are validated by negative stain transmission electron microscopy (NS-TEM). The sample is then vitrified, followed by cryo-EM data collection. Real-space helical reconstruction is performed in RELION to generate an electron potential map that is used for model building. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=37 SRC="FIGDIR/small/613698v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1b008forg.highwire.dtl.DTLVardef@c1c1e1org.highwire.dtl.DTLVardef@9fc539org.highwire.dtl.DTLVardef@14d3918_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Structure of alpha-synuclein fibrils derived from human Lewy body dementia tissue

The defining feature of Parkinson disease (PD) and Lewy body dementia (LBD) is the accumulation of alpha-synuclein (Asyn) fibrils in Lewy bodies and Lewy neurites. We developed and validated a novel method to amplify Asyn fibrils extracted from LBD postmortem tissue samples and used solid state nuclear magnetic resonance (SSNMR) studies to determine atomic resolution structure. Amplified LBD Asyn fibrils comprise two protofilaments with pseudo-21 helical screw symmetry, very low twist and an interface formed by antiparallel beta strands of residues 85-93. The fold is highly similar to the fold determined by a recent cryo-electron microscopy study for a minority population of twisted single protofilament fibrils extracted from LBD tissue. These results expand the structural landscape of LBD Asyn fibrils and inform further studies of disease mechanisms, imaging agents and therapeutics targeting Asyn.

neuroscience↗

bioRxiv2023

13C and 15N Resonance Assignments of Alpha Synuclein Fibrils Amplified from Lewy Body Dementia Tissue

Fibrils of the protein -synuclein (Asyn) are implicated in the pathogenesis of Parkinson Disease, Lewy Body Dementia, and Multiple System Atrophy. Numerous forms of Asyn fibrils have been studied by solid-state NMR and resonance assignments have been reported. Here, we report a new set of 13C, 15N assignments that are unique to fibrils obtained by amplification from postmortem brain tissue of a patient diagnosed with Lewy Body Dementia.

biophysics↗