Victoria A. Higman

TSG-6: a pluripotent inflammatory mediator?

TSG-6 is a multifunctional protein that is up-regulated in many pathological and physiological contexts, where it plays important roles in inflammation and tissue remodelling. For example, it is a potent inhibitor of neutrophil migration and can modulate the protease network through inhibition of plasmin. TSG-6 binds a wide range of GAGs (glycosaminoglycans) [i.e. HA (hyaluronan), chondroitin 4-sulphate, dermatan sulphate, heparin and heparan sulphate] as well as a variety of protein ligands, where these interactions can influence the activities of TSG-6. For example, through its association with HA, TSG-6 can mediate HA cross-linking via several different mechanisms, some of which promote leucocyte adhesion. Binding to heparin, however, enhances the ability of TSG-6 to potentiate the anti-plasmin activity of inter-alpha-inhibitor, which binds non-covalently to TSG-6 via its bikunin chain. Furthermore, although HA and heparin interact with distinct sites on the Link module, the binding of heparin can inhibit subsequent interaction with HA. In addition, the interactions of TSG-6 with HA, heparin and at least some of its protein ligands are sensitive to pH. Therefore it seems that in different tissue micro-environments (characterized, for example, by pH and GAG content), TSG-6 could be partitioned into functional pools with distinct activities.

His-384 Allotypic Variant of Factor H Associated with Age-related Macular Degeneration Has Different Heparin Binding Properties from the Non-disease-associated Form

A polymorphism in complement factor H has recently been associated with age-related macular degeneration (AMD), the leading cause of blindness in the elderly. A histidine rather than a tyrosine at residue position 384 in the mature protein increases the risk of AMD. Here, using a recombinant construct, we show that amino acid 384 is adjacent to a heparin-binding site in CCP7 of factor H and demonstrate that the allotypic variants differentially recognize heparin. This functional alteration may affect binding of factor H to polyanionic patterns on host surfaces, potentially influencing complement activation, immune complex clearance, and inflammation in the macula of AMD patients.

Rapid Proton-Detected NMR Assignment for Proteins with Fast Magic Angle Spinning

Using a set of six 1H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5–30 kDa proteins. The approach relies on perdeuteration, amide 2H/1H exchange, high magnetic fields, and high-spinning frequencies (ωr/2π ≥ 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary 13C/15N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR.

Structural diversity in the RGS domain and its interaction with heterotrimeric G protein alpha-subunits.

Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits and thus facilitate termination of signaling initiated by G protein-coupled receptors (GPCRs). RGS proteins hold great promise as disease intervention points, given their signature role as negative regulators of GPCRs—receptors to which the largest fraction of approved medications are currently directed. RGS proteins share a hallmark RGS domain that interacts most avidly with Gα when in its transition state for GTP hydrolysis; by binding and stabilizing switch regions I and II of Gα, RGS domain binding consequently accelerates Gα-mediated GTP hydrolysis. The human genome encodes more than three dozen RGS domain-containing proteins with varied Gα substrate specificities. To facilitate their exploitation as drug-discovery targets, we have taken a systematic structural biology approach toward cataloging the structural diversity present among RGS domains and identifying molecular determinants of their differential Gα selectivities. Here, we determined 14 structures derived from NMR and x-ray crystallography of members of the R4, R7, R12, and RZ subfamilies of RGS proteins, including 10 uncomplexed RGS domains and 4 RGS domain/Gα complexes. Heterogeneity observed in the structural architecture of the RGS domain, as well as in engagement of switch III and the all-helical domain of the Gα substrate, suggests that unique structural determinants specific to particular RGS protein/Gα pairings exist and could be used to achieve selective inhibition by small molecules.

Assigning large proteins in the solid state: a MAS NMR resonance assignment strategy using selectively and extensively 13C-labelled proteins.

In recent years, solid-state magic-angle spinning nuclear magnetic resonance spectroscopy (MAS NMR) has been growing into an important technique to study the structure of membrane proteins, amyloid fibrils and other protein preparations which do not form crystals or are insoluble. Currently, a key bottleneck is the assignment process due to the absence of the resolving power of proton chemical shifts. Particularly for large proteins (approximately >150 residues) it is difficult to obtain a full set of resonance assignments. In order to address this problem, we present an assignment method based upon samples prepared using [1,3-13C]- and [2-13C]-glycerol as the sole carbon source in the bacterial growth medium (so-called selectively and extensively labelled protein). Such samples give rise to higher quality spectra than uniformly [13C]-labelled protein samples, and have previously been used to obtain long-range restraints for use in structure calculations. Our method exploits the characteristic cross-peak patterns observed for the different amino acid types in 13C-13C correlation and 3D NCACX and NCOCX spectra. An in-depth analysis of the patterns and how they can be used to aid assignment is presented, using spectra of the chicken α-spectrin SH3 domain (62 residues), αB-crystallin (175 residues) and outer membrane protein G (OmpG, 281 residues) as examples. Using this procedure, over 90% of the Cα, Cβ, C′ and N resonances in the core domain of αB-crystallin and around 73% in the flanking domains could be assigned (excluding 24 residues at the extreme termini of the protein).

Structure Calculation from Unambiguous Long-Range Amide and Methyl 1H−1H Distance Restraints for a Microcrystalline Protein with MAS Solid-State NMR Spectroscopy

Magic-angle spinning (MAS) solid-state NMR becomes an increasingly important tool for the determination of structures of membrane proteins and amyloid fibrils. Extensive deuteration of the protein allows multidimensional experiments with exceptionally high sensitivity and resolution to be obtained. Here we present an experimental strategy to measure highly unambiguous spatial correlations for distances up to 13 Å. Two complementary three-dimensional experiments, or alternatively a four-dimensional experiment, yield highly unambiguous cross-peak assignments, which rely on four encoded chemical shift dimensions. Correlations to residual aliphatic protons are accessible via synchronous evolution of the (15)N and (13)C chemical shifts, which encode valuable amide-methyl distance restraints. On average, we obtain six restraints per residue. Importantly, 50% of all restraints correspond to long-range distances between residues i and j with |i - j| > 5, which are of particular importance in structure calculations. Using ARIA, we calculate a high-resolution structure for the microcrystalline 7.2 kDa α-spectrin SH3 domain with a backbone precision of ∼1.1 Å.

A software framework for analysing solid-state MAS NMR data

Solid-state magic-angle-spinning (MAS) NMR of proteins has undergone many rapid methodological developments in recent years, enabling detailed studies of protein structure, function and dynamics. Software development, however, has not kept pace with these advances and data analysis is mostly performed using tools developed for solution NMR which do not directly address solid-state specific issues. Here we present additions to the CcpNmr Analysis software package which enable easier identification of spinning side bands, straightforward analysis of double quantum spectra, automatic consideration of non-uniform labelling schemes, as well as extension of other existing features to the needs of solid-state MAS data. To underpin this, we have updated and extended the CCPN data model and experiment descriptions to include transfer types and nomenclature appropriate for solid-state NMR experiments, as well as a set of experiment prototypes covering the experiments commonly employed by solid-sate MAS protein NMR spectroscopists. This work not only improves solid-state MAS NMR data analysis but provides a platform for anyone who uses the CCPN data model for programming, data transfer, or data archival involving solid-state MAS NMR data.

Regulation of endosomal membrane traffic by a Gadkin/AP-1/kinesin KIF5 complex

Endosomes and endosomal vesicles (EVs) rapidly move along cytoskeletal filaments allowing them to exchange proteins and lipids between different endosomal compartments, lysosomes, the trans-Golgi network (TGN), and the plasma membrane. The precise mechanisms that connect membrane traffic between the TGN and perinuclear endosomal compartments with motor-protein driven transport have largely remained elusive. Here we show that Gadkin (also termed γ-BAR), a peripheral membrane protein localized to the TGN and to TGN-derived EVs, directly associates with the clathrin adaptor AP-1 and with the motor protein kinesin KIF5, thereby potentially regulating EV dynamics. Gadkin overexpression induced the dispersion of transferrin (Tf)- and Rab4-positive EVs to the cell periphery, whereas KIF5B-depleted cells displayed a perinuclear concentration. Functional experiments suggest that the role of Gadkin as a regulator of endosomal membrane traffic critically depends on complex formation with both AP-1 and KIF5. Our data thus provide a direct molecular link between TGN-derived EVs and the microtubule-based cytoskeleton.

The HicA toxin from Burkholderia pseudomallei has a role in persister cell formation

TA (toxin–antitoxin) systems are widely distributed amongst bacteria and are associated with the formation of antibiotic tolerant (persister) cells that may have involvement in chronic and recurrent disease. We show that overexpression of the Burkholderia pseudomallei HicA toxin causes growth arrest and increases the number of persister cells tolerant to ciprofloxacin or ceftazidime. Furthermore, our data show that persistence towards ciprofloxacin or ceftazidime can be differentially modulated depending on the level of induction of HicA expression. Deleting the hicAB locus from B. pseudomallei K96243 significantly reduced persister cell frequencies following exposure to ciprofloxacin, but not ceftazidime. The structure of HicA(H24A) was solved by NMR and forms a dsRBD-like (dsRNA-binding domain-like) fold, composed of a triple-stranded β-sheet, with two helices packed against one face. The surface of the protein is highly positively charged indicative of an RNA-binding protein and His24 and Gly22 were functionality important residues. This is the first study demonstrating a role for the HicAB system in bacterial persistence and the first structure of a HicA protein that has been experimentally characterized.

The Conformation of Bacteriorhodopsin Loops in Purple Membranes Resolved by Solid-State MAS NMR Spectroscopy†

Membrane proteins (and rhodopsin-like G-protein coupled receptors (GPCRs), in particular) are of significant biological and medical importance since they represent over 50% (GPCRs 25%) of current drug targets. However, the structure determination of membrane proteins is challenging: currently they account for < 1% of the unique protein structures deposited in the Protein Databank. X-ray crystallography has been used to make major contributions towards the structure determination of membrane proteins, but it suffers from the fact that the proteins are rarely crystallized in their native lipid environment or are unmodified, and exposed loop regions are often either dynamic and not visible, or involved in crystal contacts. NMR spectroscopic studies of membrane proteins in solution are generally also reliant on an artificial detergent environment and are also limited by protein size. Solid-state NMR (ssNMR) spectroscopy, in contrast, has the advantage that membrane proteins can be studied in a lipid environment. Although ssNMR does not suffer from the same intrinsic size limitation as solution NMR spectroscopy, spectral overlap is often severe for large proteins and hampers their study. However, magicangle-spinning (MAS) NMR spectroscopy, in particular, has been used to make substantial methodological advances in recent years, and the first membrane protein structures have now been determined using this technique. Herein we report on how solid-state MAS NMR spectroscopy can be used to complement X-ray crystallographic studies of a large seven transmembrane (7TM) helical protein by validating and redefining the loop structures. The structure of bacteriorhodopsin (bR) has been determined in a range of two(2D) and three-dimensional (3D) crystalline environments with the loops showing the highest degree of structural variation. Solid-state MAS NMR spectra of uniformly [C,N]-labeled bR in its native purple membrane have been used to assign the signals of the loop regions of the protein. Extraction of dihedral angle information from chemical shifts has allowed us to validate several loop conformations in the crystal structure and recalculate the structure where there are differences in conformation. Ab initio assignment of the resonances of the loop regions of bR was carried out using 2D DARR spectra (mixing times of 15 and 50 ms) and 3D NCACX (20 ms), 3D NCOCX (20 ms), 3D CANCO and 3D CAN(CO)CX (45 ms) spectra. Assignment of the loops is made possible by the fact that the loop resonances are generally well separated and amenable to assignment in contrast to many of the helical regions, where leucine and valine resonances, in particular, exhibit intractable degrees of spectral overlap. Figure 1 shows the assignment of the section Met68–Gly72 in the BC loop as an example; further 2D spectra and strip plots are provided in the Supporting Information (Figures S1–S3). In total, we have assigned roughly 55% of loop residues covering all loops, except for the CD loop, as well as several residues located in the helices (Figure 2, Table S2 in the Supporting Information, and BMRB Accession code 17361). Interestingly, residues from all loops (except those in the unassigned CD loop) are visible in our cross-polarization (CP)-based spectra; this is in contrast to the spectra of sensory rhodopsin II from Natronomonas pharaonis (NpSRII) where most loops were visible only in INEPT-based spectra, reflecting a higher degree of loop mobility in NpSRII. Our observations are more similar to those made in a recent study of proteorhodopsin in which only isolated residues were observed in INEPT-based spectra. A C,C INEPT-COSY spectrum of bR contains resonances with random-coil chemical shifts from amino acid types that are consistent with the Nand C-terminal tails (see Figures S4 and S5 in Supporting Information). Some chemical shifts for side chains in non-random-coil conformations are also found for residues Lys, Glu, Ala, and Ser, which may belong to the KAES motif in the EF loop. Sequential [*] Dr. V. A. Higman, Dr. P. J. Judge, Prof. A. Watts Department of Biochemistry, University of Oxford South Parks Road, Oxford, OX1 3QU (UK) E-mail: anthony.watts@bioch.ox.ac.uk Dr. K. Varga Department of Chemistry, University of Wyoming Laramie, WY 82071 (USA)