An approach combining small-angle X-ray solution scattering (SAXS) data with coarse-grained (CG) simulations is developed to characterize the assembly states of Hck, a member of the Src-family ...kinases, under various conditions in solution. First, a basis set comprising a small number of assembly states is generated from extensive CG simulations. Second, a theoretical SAXS profile for each state in the basis set is computed by using the Fast-SAXS method. Finally, the relative population of the different assembly states is determined via a Bayesian-based Monte Carlo procedure seeking to optimize the theoretical scattering profiles against experimental SAXS data. The study establishes the concept of basis-set supported SAXS (BSS-SAXS) reconstruction combining computational and experimental techniques. Here, BSS-SAXS reconstruction is used to reveal the structural organization of Hck in solution and the different shifts in the equilibrium population of assembly states upon the binding of different signaling peptides.
Although ACE2 (angiotensin converting enzyme 2) is considered the primary receptor for CoV-2 cell entry, recent reports suggest that alternative pathways may contribute. This paper considers the ...hypothesis that viral binding to cell-surface integrins may contribute to the high infectivity and widespread extra-pulmonary impacts of the SARS-CoV-2 virus. This potential is suggested on the basis of the emergence of an RGD (arginine-glycine-aspartate) sequence in the receptor-binding domain of the spike protein. RGD is a motif commonly used by viruses to bind cell-surface integrins. Numerous signaling pathways are mediated by integrins and virion binding could lead to dysregulation of these pathways, with consequent tissue damage. Integrins on the surfaces of pneumocytes, endothelial cells and platelets may be vulnerable to CoV-2 virion binding. For instance, binding of intact virions to integrins on alveolar cells could enhance viral entry. Binding of virions to integrins on endothelial cells could activate angiogenic cell signaling pathways; dysregulate integrin-mediated signaling pathways controlling developmental processes; and precipitate endothelial activation to initiate blood clotting. Such a procoagulant state, perhaps together with enhancement of platelet aggregation through virions binding to integrins on platelets, could amplify the production of microthrombi that pose the threat of pulmonary thrombosis and embolism, strokes and other thrombotic consequences. The susceptibility of different tissues to virion-integrin interactions may be modulated by a host of factors, including the conformation of relevant integrins and the impact of the tissue microenvironment on spike protein conformation. Patient-specific differences in these factors may contribute to the high variability of clinical presentation. There is danger that the emergence of receptor-binding domain mutations that increase infectivity may also enhance access of the RGD motif for integrin binding, resulting in viral strains with ACE2 independent routes of cell entry and novel integrin-mediated biological and clinical impacts. The highly infectious variant, B.1.1.7 (or VUI 202012/01), includes a receptor-binding domain amino acid replacement, N501Y, that could potentially provide the RGD motif with enhanced access to cell-surface integrins, with consequent clinical impacts.
Donald Caspar (1927-2021) Makowski, Lee
Nature (London),
01/2022, Letnik:
601, Številka:
7891
Journal Article
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The same rules are also essential in designing viral vectors to deliver gene therapy. Caspar used limited data from X-ray crystallography, electron microscopy and fibre diffraction to tease out ...features of prototypical viruses. In 1958, Caspar established what would become one of the most influential centres for the study of macromolecular structure: the Laboratory for Structural Biology at the Children's Cancer Research Foundation at Boston Children's Hospital.
We present a coarse residue-based computational method to rapidly compute the solution scattering profile from a protein with dynamical fluctuations. The method is built upon a coarse-grained (CG) ...representation of the protein. This CG representation takes advantage of the intrinsic low-resolution and CG nature of solution scattering data. It allows rapid scattering determination from a large number of conformations that can be extracted from CG simulations to obtain scattering characterization of protein conformations. The method includes several important elements, effective residue structure factors derived from the Protein Data Bank, explicit treatment of water molecules in the hydration layer at the surface of the protein, and an ensemble average of scattering from a variety of appropriate conformations to account for macromolecular flexibility. This simplified method is calibrated and illustrated to accurately reproduce the experimental scattering curve of Hen egg white lysozyme. We then illustrated the applications of this CG method by computing the solution scattering patterns of several representative protein folds and multiple conformational states. The results suggest that solution scattering data, when combined with the reliable computational method that we developed, show great potential for a better structural description of multidomain complexes in different functional states, and for recognizing structural folds when sequence similarity to a protein of known structure is low.
Characterization of immunoglobulin solutions at high concentrations represents a significant challenge. A current trend in the biopharmaceutical industry is to manufacture highly concentrated drug ...products, which can be used to deliver high doses in small volumes, via subcutaneous injections. Studying a molecule's structure and properties in its final drug product formulation is ideal, but characterization is typically performed under dilute solution conditions with critical stabilizing buffer components removed because of interference effects, which can result in an incomplete understanding of the molecule's properties. Direct study of protein conformation and protein-protein interactions in concentrated solutions is challenging for most biophysical and biochemical techniques; however, X-ray solution scattering offers opportunities. Combined with other biophysical techniques, X-ray scattering has the potential to provide relevant information on both structure and interactions in protein solutions over a broad concentration range. Here, we report X-ray solution scattering of 4 monoclonal antibodies, designated mAb1 (glycosylated and de-glycosylated), mAb2, and mAb3 at concentrations between 0.5 and >168 mg/mL. Data acquired from these measurements are combined with the results from other biophysical measurements to generate a comprehensive profile of their solution behaviors. Our results show that X-ray solution scattering can assess key parameters needed to aid in formulation development.
This paper describes a computational approach to estimating wide‐angle X‐ray solution scattering (WAXS) from proteins, which has been implemented in a computer program called SoftWAXS. The accuracy ...and efficiency of SoftWAXS are analyzed for analytically solvable model problems as well as for proteins. Key features of the approach include a numerical procedure for performing the required spherical averaging and explicit representation of the solute–solvent boundary and the surface of the hydration layer. These features allow the Fourier transform of the excluded volume and hydration layer to be computed directly and with high accuracy. This approach will allow future investigation of different treatments of the electron density in the hydration shell. Numerical results illustrate the differences between this approach to modeling the excluded volume and a widely used model that treats the excluded‐volume function as a sum of Gaussians representing the individual atomic excluded volumes. Comparison of the results obtained here with those from explicit‐solvent molecular dynamics clarifies shortcomings inherent to the representation of solvent as a time‐averaged electron‐density profile. In addition, an assessment is made of how the calculated scattering patterns depend on input parameters such as the solute‐atom radii, the width of the hydration shell and the hydration‐layer contrast. These results suggest that obtaining predictive calculations of high‐resolution WAXS patterns may require sophisticated treatments of solvent.
Aggregation of Aβ amyloid fibrils into plaques in the brain is a universal hallmark of Alzheimer's Disease (AD), but whether plaques in different individuals are equivalent is unknown. One ...possibility is that amyloid fibrils exhibit different structures and different structures may contribute differentially to disease, either within an individual brain or between individuals. However, the occurrence and distribution of structural polymorphisms of amyloid in human brain is poorly documented. Here we use X-ray microdiffraction of histological sections of human tissue to map the abundance, orientation and structural heterogeneities of amyloid. Our observations indicate that (i) tissue derived from subjects with different clinical histories may contain different ensembles of fibrillar structures; (ii) plaques harboring distinct amyloid structures can coexist within a single tissue section and (iii) within individual plaques there is a gradient of fibrillar structure from core to margins. These observations have immediate implications for existing theories on the inception and progression of AD.
Alzheimer's disease (AD) is a neurodegenerative disorder defined by the progressive formation and spread of fibrillar aggregates of Aβ peptide and tau protein. Polymorphic forms of these aggregates ...may contribute to disease in varying ways since different neuropathologies appear to be associated with different sets of fibrillar structures and follow distinct pathological trajectories that elicit characteristic clinical phenotypes. The molecular mechanisms underlying the spread of these aggregates in disease may include nucleation, replication, and migration all of which could vary with polymorphic form, stage of disease, and region of brain. Given the linkage between mechanisms of progression and distribution of polymorphs, mapping the distribution of fibrillar structures
has the potential to discriminate between mechanisms of progression. However, the means of carrying out this mapping are limited. Optical microscopy lacks the resolution to discriminate between polymorphs
, and higher resolution tools such as ssNMR and cryoEM require the isolation of fibrils from tissue, destroying relevant spatial information. Here, we demonstrate the use of scanning x-ray microdiffraction (XMD) to map the locations of fibrillar polymorphs of Aβ peptides and tau protein in histological thin sections of human brain tissue. Coordinated examination of serial sections by immunohistochemistry was used to aid in the interpretation of scattering patterns and to put the observations in a broader anatomical context. Scattering from lesions in tissue shown to be rich in Aβ fibrils by immunohistochemistry exhibited scattering patterns with a prototypical 4.7 Å cross-β peak, and overall intensity distribution that compared well with that predicted from high resolution structures. Scattering from lesions in tissue with extensive tau pathology also exhibited a 4.7 Å cross-β peak but with intensity distributions that were distinct from those seen in Aβ-rich regions. In summary, these observations demonstrate that XMD is a rich source of information on the distribution of fibrillar polymorphs in diseased human brain tissue. When used in coordination with neuropathological examination it has the potential to provide novel insights into the molecular mechanisms underlying disease.
Scanning x-ray microdiffraction of complex tissues and materials is an emerging method for the study of macromolecular structures in situ, providing information on the way molecular constituents are ...arranged and interact with their microenvironment. Acting as a bridge between high-resolution images of individual constituents and lower resolution microscopies that generate global views of material, scanning microdiffraction provides an approach to study the functioning of complex tissues across multiple length scales. Here, we discuss the methodology, summarize results from recent studies, and discuss the potential of the technique for future studies coordinated with other biophysical techniques.
Understanding how enzymes achieve their tremendous catalytic power is a major question in biochemistry. Greater understanding is also needed for enzyme engineering applications. In many cases, enzyme ...efficiency and specificity depend on residues not in direct contact with the substrate, termed remote residues. This work focuses on Escherichia coli ornithine transcarbamoylase (OTC), which plays a central role in amino acid metabolism. OTC has been reported to undergo an induced-fit conformational change upon binding its first substrate, carbamoyl phosphate (CP), and several residues important for activity have been identified. Using computational methods based on the computed chemical properties from theoretical titration curves, sequence-based scores derived from evolutionary history, and protein surface topology, residues important for catalytic activity were predicted. The roles of these residues in OTC activity were tested by constructing mutations at predicted positions, followed by steady-state kinetics assays and substrate binding studies with the variants. First-layer mutations R57A and D231A, second-layer mutation H272L, and third-layer mutation E299Q, result in 57- to 450-fold reductions in kcat/KM with respect to CP and 44- to 580-fold reductions with respect to ornithine. Second-layer mutations D140N and Y160S also reduce activity with respect to ornithine. Most variants had decreased stability relative to wild-type OTC, with variants H272L, H272N, and E299Q having the greatest decreases. Variants H272L, E299Q, and R57A also show compromised CP binding. In addition to direct effects on catalytic activity, effects on overall protein stability and substrate binding were observed that reveal the intricacies of how these residues contribute to catalysis.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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