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Abstract: The Streptococcal C5a peptidase (ScpA) specifically inactivates the human complement factor hC5a, a potent anaphylatoxin recently identified as a therapeutic target for treatment of COVID-19 infections. Engineering of ScpA to enhance its potential as a therapeutic will require detailed examination of the basis for its highly selective activity. The emerging view of ScpA and related subtilases is that selection of their substrates is a dynamic two-step process involving flexibility in the domains around the active site and in the C-ter of the substrate. Surface plasmon resonance (SPR) analyses of the ScpA-hC5a interaction have shown that high affinity binding of the substrate is driven by electrostatic interactions between an exosite on the Fn2 domain of the enzyme and the bulky N-ter cleavage product (PN, ’core’ residues 1-67) of C5a [1]. Introduction of a D783A mutation in the Fn2 exosite, located approximately 50 Å from the catalytic serine, was shown to significantly reduce substrate binding affinity and kcat of the enzyme. X-ray crystallographic studies on the D783A mutant (ScpAD783A) were undertaken to better interpret the impact of this mutation on the specificity and activity of ScpA. Here we present the 1.9 Å X-ray diffraction data for ScpAD783A and the molecular replacement solution for the structure. Both raw diffraction images and coordinates have been made available on public databases. Additional details on the related SPR and enzyme kinetics analyses on ScpAD783A reported in Jain et al. [2].

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Abstract: Channels and transporters are essential proteins for the control, mediation and termination of neurotransmission. These are implicated in numerous pathological conditions, including epilepsy, Parkinson’s disease, neuropathic pain and nicotine addiction. However, the structural and ligand binding aspects of many of these channel and transporter proteins are poorly defined, which limits being able to design new molecules that can effectively target these conditions. It was aimed to investigate structure and ligand binding at neurotransmitter channels and transporters. The first aims involved elucidating the binding modes and structure-activity relationships of novel ligands at nicotinic acetylcholine receptors (nAChRs), using the surrogate protein acetylcholine binding protein (AChBP). The ligands being characterised were of interest as potential anti-smoking agents and as research tools for studying nAChRs. Binding data and protein complex crystal structures were obtained for several of these novel ligands and it was possible to identify residues which were potentially responsible for their modes of action and affinity to AChBP, and henceforth likely to nAChRs. Knowledge of these interactions could assist in the future design of other ligands targeting nAChRs. The second set of aims were associated with attempting to establish methodologies for the efficient recombinant production of complex eukaryotic ion channels and neurotransmitter sodium symporters. The initial objective was the insect ligand gated ion channel resistance to dieldrin (RDL), which is a target for insecticides. Sf9 insect cells proved unsuitable for production as only a small amount of the total protein could be extracted with non-ionic detergents and it was implied that the majority of the protein was likely in an un-folded state. Mammalian HEK293 cells were more successful as the protein could be efficiently solubilised, but ultimately the yields of purified protein were too low for this to be a feasible approach. There was more success with producing the human GABA transporter 1 (GAT1). This terminates the actions of the inhibitory neurotransmitter GABA by removing it from the synapse and is a therapeutic target for the control of epilepsy. Using Sf9 cells and a conventional baculovirus system showed initial success, but there were ultimately problems with aggregation. Use of a recently described baculovirus system with an early Drosophila Hsp70 promoter however resolved these problems and led to high yields of purified GAT1. The obtained protein was suggested to be potentially suitable for future structural studies by single particle cryogenic electron microscopy (cryo-EM). Purified GAT1 was also used as a target to isolate recombinant nanobodies from a yeast library and these may be of assistance for increasing the size of the protein for cryo-EM studies.

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Abstract: This thesis reports on the results of a series of crystallographic studies performed using ultrafast electron diffraction (UED) and serial X-ray crystallography on semiconductor nanocrystal and protein samples. In the first part of the thesis, ultrabright femtosecond electron pulses generated using a radio frequency-compressed photocathode electron source are employed to measure the photoinduced lattice response of lead sulfide (PbS) colloidal quantum dots following the optical generation of above-bandgap excitons. Short-range nonthermal lattice distortions are observed on the picosecond timescale in the form of exaggerated atomic disorder in the (100) crystallographic directions and deviations from a purely thermal response at short distance scales (< 12 Å). These structural changes are found to be related to surface charge trapping through size- and surface-series experiments and a pair distribution function analysis. In the second part of the thesis, a description of a new experimental scheme for serial X-ray crystallography is presented. The recent advent of free-electron lasers (XFEL) in the hard X-ray wavelength range (< 1 Å) has necessitated the use of serial femtosecond crystallography (SFX) for static and time-resolved structure determination. This has driven the innovation of novel sample delivery techniques to meet the stringent demands imposed by the SFX approach. This thesis outlines the development and implementation of a versatile high-speed fixed-target sample delivery technique based on a microfabricated silicon chip and spectroscopic sample mapping capability for use at both XFEL and synchrotron light sources. Application of the “serial crystallography chip” method maximizes hit-rates and minimizes sample consumption, as demonstrated by crystallographic experiments on the proteins: carboxy-myoglobin, lysozyme and fluoroacetate dehalogenase. An extension of the method which combines fixed-target serial crystallography with conventional rotation crystallography is also presented, which allows for X-ray induced damage-free structures to be obtained using serial crystallography at synchrotron light sources.

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Abstract: Targeted protein degradation offers an alternative modality to classical inhibition and holds the promise of addressing previously undruggable targets to provide novel therapeutic options for patients. Heterobifunctional molecules co-recruit a target protein and an E3 ligase, resulting in ubiquitylation and proteosome-dependent degradation of the target. In the clinic, the oral route of administration is the option of choice but has only been achieved so far by CRBN- recruiting bifunctional degrader molecules. We aimed to achieve orally bioavailable molecules that selectively degrade the BAF Chromatin Remodelling complex ATPase SMARCA2 over its closely related paralogue SMARCA4, to allow in vivo evaluation of the synthetic lethality concept of SMARCA2 dependency in SMARCA4-deficient cancers. Here we outline structure- and property-guided approaches that led to orally bioavailable VHL-recruiting degraders. Our tool compound, ACBI2, shows selective degradation of SMARCA2 over SMARCA4 in ex vivo human whole blood assays and in vivo efficacy in SMARCA4-deficient cancer models. This study demonstrates the feasibility for broadening the E3 ligase and physicochemical space that can be utilised for achieving oral efficacy with bifunctional molecules.

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Abstract: The Na+,K+-ATPase generates electrochemical gradients of Na+ and K+ across the plasma membrane via a functional cycle that includes various phosphoenzyme intermediates. However, the structure and function of these intermediates and how metal fluorides mimick them require further investigation. Here, we describe a 4.0 Å resolution crystal structure and functional properties of the pig kidney Na+,K+-ATPase stabilized by the inhibitor beryllium fluoride (denoted E2–BeFx). E2–BeFx is expected to mimic properties of the E2P phosphoenzyme, yet with unknown characteristics of ion and ligand binding. The structure resembles the E2P form obtained by phosphorylation from inorganic phosphate (Pi) and stabilized by cardiotonic steroids, including a low-affinity Mg2+ site near ion binding site II. Our anomalous Fourier analysis of the crystals soaked in Rb+ (a K+ congener) followed by a low-resolution rigid-body refinement (6.9–7.5 Å) revealed preocclusion transitions leading to activation of the dephosphorylation reaction. We show that the Mg2+ location indicates a site of initial K+ recognition and acceptance upon binding to the outward-open E2P state after Na+ release. Furthermore, using binding and activity studies, we find that the BeFx-inhibited enzyme is also able to bind ADP/ATP and Na+. These results relate the E2–BeFx complex to a transient K+- and ADP-sensitive E∗P intermediate of the functional cycle of the Na+,K+-ATPase, prior to E2P.