Dynamic protein gradients are exploited for the spatial organization and segregation of replicated chromosomes. However, mechanisms of protein gradient formation and how that spatially organizes chromosomes remain poorly understood. Here, we have determined the kinetic principles of subcellular localizations of ParA2 ATPase, an essential spatial regulator of chromosome 2 segregation in the multichromosome bacterium, Vibrio cholerae. We found that ParA2 gradients self-organize in V. cholerae cells into dynamic pole-to-pole oscillations. We examined the ParA2 ATPase cycle and ParA2 interactions with ParB2 and DNA. In vitro, ParA2-ATP dimers undergo a rate-limiting conformational switch, catalysed by DNA to achieve DNA-binding competence. This active ParA2 state loads onto DNA cooperatively as higher order oligomers. Our results indicate that the midcell localization of ParB2-parS2 complexes stimulate ATP hydrolysis and ParA2 release from the nucleoid, generating an asymmetric ParA2 gradient with maximal concentration toward the poles. This rapid dissociation coupled with slow nucleotide exchange and conformational switch provides for a temporal lag that allows the redistribution of ParA2 to the opposite pole for nucleoid reattachment. Based on our data, we propose a 'Tug-of-war' model that uses dynamic oscillations of ParA2 to spatially regulate symmetric segregation and positioning of bacterial chromosomes.

The cleavage of septal peptidoglycan at the end of cell division facilitates the separation of the two daughter cells. The hydrolases involved in this process (called autolysins) are potentially lethal enzymes that can cause cell death; their activity, therefore, must be tightly controlled during cell growth. In Enterococcus faecalis, the N-acetylglucosaminidase AtlA plays a predominant role in cell separation. atlA mutants form long cell chains and are significantly less virulent in the zebrafish model of infection. The attenuated virulence of atlA mutants is underpinned by a limited dissemination of bacterial chains in the host organism and a more efficient uptake by phagocytes that clear the infection. AtlA has structural homologs in other important pathogens, such as Listeria monocytogenes and Salmonella typhimurium, and therefore represents an attractive model to design new inhibitors of bacterial pathogenesis. Here, we provide a 1.45 & Aring; crystal structure of the E. faecalis AtlA catalytic domain that reveals a closed conformation of a conserved beta-hairpin and a complex network of hydrogen bonds that bring two catalytic residues to the ideal distance for an inverting mechanism. Based on the model of the AtlA-substrate complex, we identify key residues critical for substrate recognition and septum cleavage during bacterial growth. We propose that this work will provide useful information for the rational design of specific inhibitors targeting this enterococcal virulence factor and its orthologs in other pathogens.

Self-assembly of the amyloid-β (Aβ) peptide to form toxic oligomers and fibrils is a key causal event in the onset of Alzheimer's disease, and Aβ is the focus of intense research in neuroscience, biophysics, and structural biology aimed at therapeutic development. Due to its rapid self-assembly and extreme sensitivity to aggregation conditions, preparation of seedless, reproducible Aβ solutions is highly challenging, and there are serious ongoing issues with consistency in the literature. In this paper, we use a liquid-phase separation technique, asymmetric flow field-flow fractionation with multiangle light scattering (AF4-MALS), to develop and validate a simple, effective, economical method for re-solubilization and quality control of purified, lyophilized Aβ samples. Our findings were obtained with recombinant peptide but are physicochemical in nature and thus highly relevant to synthetic peptide. We show that much of the variability in the literature stems from the inability of overly mild solvent treatments to produce consistently monomeric preparations and is rectified by a protocol involving high-pH (>12) dissolution, sonication, and rapid freezing to prevent modification. Aβ treated in this manner is chemically stable, can be stored over long timescales at -80 °C, and exhibits remarkably consistent self-assembly behavior when returned to near-neutral pH. These preparations are highly monomeric, seedless, and do not require additional rounds of size exclusion, eliminating the need for this costly procedure and increasing the flexibility of use. We propose that our improved protocol is the simplest, fastest, and most effective way to solubilize Aβ from diverse sources for sensitive self-assembly and toxicity assays.

ABSTRACT Vibrio cholerae chromosome 2 (Chr2) requires its own ParABS system for segregation. Without it, V. cholerae becomes nonviable and loses pathogenicity. ParA2 of Chr2 is a Walker-type ATPase that is the main driver of Chr2 segregation. Most of our understanding of ParA function comes from studying plasmid partition systems. How ParA provides the motive force in segregation of chromosomes, which are much larger than plasmids, is less understood and different models have been proposed. Here we analyzed in vivo behavior and kinetic properties of ParA2 using cell imaging, biochemical and biophysical approaches. ParA2 formed an asymmetric gradient in the cell that localized dynamically in the cell cycle. We found that ParA2 dimers bind ATP and undergo a slow conformational change to an active DNA-binding state, similar to P1 ParA. The presence of DNA catalyzes ParA2 conformational change to allow cooperative binding of active ParA2 dimers to form higher-order oligomers on DNA. Nucleotide exchange rates were also slow, thus providing a control of ParA2 recruitment and dynamic localizations. Although highly conserved in biochemical properties, ParA2 showed faster overall ATP cycling and DNA-rebinding rates than plasmid ParAs, suggesting that this could be shared kinetic features among chromosomal ParAs to regulate the transport of a much larger DNA cargo.

High-concentration (>100 g/L) solutions of monoclonal antibodies (mAbs) are typically characterized by anomalously large solution viscosity and shear thinning behavior for strain rates ≥103 s-1. Here, the link between protein-protein interactions (PPIs) and the rheology of concentrated solutions of COE-03 and COE-19 mAbs is studied by means of static and dynamic light scattering and microfluidic rheometry. By comparing the experimental data with predictions based on the Baxter sticky hard-sphere model, we surprisingly find a connection between the observed shear thinning and the predicted percolation threshold. The longest shear relaxation time of mAbs was much larger than that of model sticky hard spheres within the same region of the phase diagram, which is attributed to the anisotropy of the mAb PPIs. Our results suggest that not only the strength but also the patchiness of short-range attractive PPIs should be explicitly accounted for by theoretical approaches aimed at predicting the shear rate-dependent viscosity of dense mAb solutions.

Molecular crowding in highly concentrated monoclonal antibody (mAb) solutions results in significant increases in viscosity, which complicates fill-finish steps and patient administration by subcutaneous injection. As viscosity measurements for optimization of the mAb formulation require significant amounts of material not always available in early development, fluorescence correlation spectroscopy (FCS) is evaluated as a potential ultra-low volume technique for viscosity measurement of high concentration protein solutions assuming the Generalised Stokes Einstein relation (GSE) remains valid. Using like-charge fluorescent tracers of different sizes, FCS provided measurements of microviscosities which were compared to the macroviscosity. After parametrising the protein concentration dependence of the viscosity by the exponential coefficient (k) of a simple exponential model, FCS derived k-values of like-size tracer to the crowder followed the same ordering as the macroviscosity derived k-values with respect to solvent conditions. Furthermore, k and the diffusion-derived protein-protein interaction parameter, kD, are linked, and, attractive conditions for mAbs result in a stronger concentration dependence of the viscosity. For tracers and crowders of like-size, a key result is negative deviations from the GSE relation are observed in presence of strong attractive interactions between crowder molecules. These data demonstrate that FCS has application to the screening of high concentration mAb solutions for formulation selection.

3D domain-swapping proteins form multimers by unfolding and then sharing of secondary structure elements, often with native-like interactions. Runaway domain swapping is proposed as a mechanism for folded proteins to form amyloid fibres, with examples including serpins and cystatins. Cystatin C amyloids cause a hereditary form of cerebral amyloid angiopathy whilst cystatin B aggregates are found in cases of Unverricht-Lundborg Syndrome, a progressive form of myoclonic epilepsy. Under conditions that favour fibrillisation, cystatins populate stable 3D domain-swapped dimers both in vitro and in vivo that represent intermediates on route to the formation of fibrils. Previous work on cystatin B amyloid fibrils revealed that the α-helical region of the protein becomes disordered and identified the conservation of a continuous 20-residue elongated β-strand (residues 39-58), the latter being a salient feature of the dimeric 3D domain-swapped structure. Here we apply limited proteolysis to cystatin B amyloid fibrils and show that not only the α-helical N-terminal of the protein (residues 1-35) but also the C-terminal of the protein (residues 80-98) can be removed without disturbing the underlying fibril structure. This observation is incompatible with previous models of cystatin amyloid fibrils where the β-sheet is assumed to retain its native antiparallel arrangement. We conclude that our data favour a more generic, at least partially parallel, arrangement for cystatin β-sheet structure in mature amyloids and propose a model that remains consistent with available data for amyloids from either cystatin B or cystatin C.

Misfolded protein aggregates, characterized by a canonical amyloid fold, play a central role in the pathobiology of neurodegenerative diseases. Agents that bind and sequester neurotoxic intermediates of amyloid assembly, inhibit the assembly or promote the destabilization of such protein aggregates are in clinical testing. Here, we show that the gene 3 protein (g3p) of filamentous bacteriophage mediates potent generic binding to the amyloid fold. We have characterized the amyloid binding and conformational remodeling activities using an array of techniques, including X-ray fiber diffraction and NMR. The mechanism for g3p binding with amyloid appears to reflect its physiological role during infection of Escherichia coli, which is dependent on temperature-sensitive interdomain unfolding and cis–trans prolyl isomerization of g3p. In addition, a natural receptor for g3p, TolA-C, competitively interferes with Aβ binding to g3p. NMR studies show that g3p binding to Aβ fibers is predominantly through middle and C-terminal residues of the Aβ subunit, indicating β strand–g3p interactions. A recombinant bivalent g3p molecule, an immunoglobulin Fc (Ig) fusion of the two N-terminal g3p domains, (1) potently binds Aβ fibers (fAβ) (KD=9.4nM); (2); blocks fAβ assembly (IC50~50nM) and (3) dissociates fAβ (EC50=40–100nM). The binding of g3p to misfolded protein assemblies is generic, and amyloid-targeted activities can be demonstrated using other misfolded protein systems. Taken together, our studies show that g3p(N1N2) acts as a general amyloid interaction motif.

Protein misfolding that produces assemblies of toxic and transmissible aggregates is a central feature of the pathobiology of neurodegenerative diseases. A serendipitous discovery that direct exposure to filamentous bacteriophage M13 mediates reductions of both Aβ and tau deposits in brains of transgenic mouse models of Alzheimer's disease led to a search for the mechanism. We have isolated and characterized a fragment of the phage capsid protein responsible for the amyloid targeting activities of M13, and we show that an immunoglobulin fusion of this motif (Ig-GAIM) recapitulates the efficacy of M13 both in vitro and in transgenic Alzheimer's disease model mice following either intrahippocampal or chronic systemic administration. SPR, ELISA and dot blot based assays were used for binding analysis of fibers and oligomers. Fibers remodeling was shown by filter retention assay. Fiber assembly inhibition was monitored using ThT binding assays and transmission electron microscopy. Nuclear magnetic resonance spectroscopy was used to map binding of GAIM to Aβ fibers using H/D exchange technique. Blocking of oligomer-Induced cytotoxicity using Ig-GAIM is shown as well. Efficacy in an aged Tg2576 AD mouse model was measured by Aβ ELISA and immunohistochemical Aβ plaque load in the hippocampus 7 days after a single bilateral intracranial injection of Ig-GAIM. Assays for amyloid fiber binding and remodeling, fiber assembly inhibition, and neuroprotection from cytotoxic oligomers together suggest this protein motif mediates these activities by potently preventing edge-to-edge beta strand aggregation. We also show that the GAIM activity mechanism is highly related to the natural role of bacteriophage infection mechanism. This targeting hypothesis is further substantiated by NMR H/D exchange spectroscopy of complexes of fibrilar Aβ and the capsid protein and by binding specificity studies on an array of misfolded and natively folded proteins. Collectively, these results indicate on a novel general mechanism of amyloid specific recognition, disruption and a potentially broad therapeutic modality. Systemic administration of GAIM Ig fusion reverses behavioral, biochemical and neuropathologic endpoints in both Aβ and Tau mouse models. The Ig-GAIM fusions represent a novel, potent, and specific therapeutic approach for reducing pathologic misfolded protein assemblies that are central players in neurodegenerative diseases.

Amyloid fibres are characteristic of over 25 degenerative human diseases including Alzheimer’s and Parkinson’s disease. Amyloid fibres are insoluble, highly stable, ordered cross-β sheet structures, which form as a result of conformational change and aggregation in a range of unrelated soluble proteins and peptides. High molecular weight oligomers, potentially on-pathway to amyloid formation, have been posited as the toxicity agent in amyloid-associated disease. Heterogeneity of amyloid fibres and oligomers precludes the use of standard structural biology techniques. The work presented utilised recombinant human cystatin B as a model system for structural analysis of amyloid fibres and oligomers, particularly for the structurally homologous human cystatin C which directly causes a form of amyloid angiopathy. Limited proteolysis of cystatin B amyloid fibre shows that the core structure consists maximally of residues 27-80 out of a total 98. Electron microscopy techniques, including mass per unit length measurements, reveal an average fibre width of 8.6 nm and formation of four fibre classes, composed of 4, 8 and 16 molecules per 4 -strand rise. These data are incompatible with the previous native-like model, therefore a new working fibre model is proposed where native β-strands 2 and 3 are extended into a single strand with adjacent β-strands 4 and 5 forming the other half of a β-sheet arc, which is stacked in parallel, perpendicular to the fibre axis. Stable proteinase-K resistant G4R mutant cystatin B oligomers were generated and preliminary characterisation presented, with identification of five structural classes ranging in diameter from 8-88 nm. Furthermore, investigation of a potential amyloid therapeutic in Salvia plant extracts was carried out with dye binding kinetic assays and electron microscopy. The work herein primarily constrains a new non-native β-sheet cystatin B amyloid fibre model and has initiated new avenues of research into cystatin oligomer structure and novel therapeutic agents.

Unlike a number of amyloid-forming proteins, stefins, and in particular stefin B (cystatin B) form amyloids under conditions where the native state predominates. In order to trigger oligomerization processes, the stability of the protein needs to be compromised, favoring structural re-arrangement however, accelerating fibril formation is not a simple function of protein stability. We report here on how optimal conditions for amyloid formation lead to the destabilization of dimeric and tetrameric states of the protein in favor of the monomer. Small, highly localized structural changes can be mapped out that allow us to visualize directly areas of the protein which eventually become responsible for triggering amyloid formation. These regions of the protein overlap with the Cu (II)-binding sites which we identify here for the first time. We hypothesize that in vivo modulators of amyloid formation may act similarly to painstakingly optimized solvent conditions developed in vitro. We discuss these data in the light of current structural models of stefin B amyloid fibrils based on H-exchange data, where the detachment of the helical part and the extension of loops were observed.

It is well established that contact order and folding rates are correlated for small proteins. The folding rates of stefins A and B differ by nearly two orders of magnitude despite sharing an identical native fold and hence contact order. We break down the determinants of this behavior and demonstrate that the modulation of contact order effects can be accounted for by the combined contributions of a framework-like mechanism, characterized by intrinsic helix stabilities, together with nonnative helical backbone conformation and nonnative hydrophobic interactions within the folding transition state. These contributions result in the formation of nonnative interactions in the transition state as evidenced by the opposing effects on folding rate and stability of these proteins.