A novel approach is presented for an a priori assessment of the ambiguity associated with spherically averaged single‐particle scattering. The approach is of broad interest to the structural biology ...community, allowing the rapid and model‐independent assessment of the inherent non‐uniqueness of three‐dimensional shape reconstruction from scattering experiments on solutions of biological macromolecules. One‐dimensional scattering curves recorded from monodisperse systems are nowadays routinely utilized to generate low‐resolution particle shapes, but the potential ambiguity of such reconstructions remains a major issue. At present, the (non)uniqueness can only be assessed by a posteriori comparison and averaging of repetitive Monte Carlo‐based shape‐determination runs. The new a priori ambiguity measure is based on the number of distinct shape categories compatible with a given data set. For this purpose, a comprehensive library of over 14 000 shape topologies has been generated containing up to seven beads closely packed on a hexagonal grid. The computed scattering curves rescaled to keep only the shape topology rather than the overall size information provide a `scattering map' of this set of shapes. For a given scattering data set, one rapidly obtains the number of neighbours in the map and the associated shape topologies such that in addition to providing a quantitative ambiguity measure the algorithm may also serve as an alternative shape‐analysis tool. The approach has been validated in model calculations on geometrical bodies and its usefulness is further demonstrated on a number of experimental X‐ray scattering data sets from proteins in solution. A quantitative ambiguity score (a‐score) is introduced to provide immediate and convenient guidance to the user on the uniqueness of the ab initio shape reconstruction from the given data set.
Small‐angle X‐ray scattering (SAXS) is one of the major tools for the study of model membranes, but interpretation of the scattering data remains non‐trivial. Current approaches allow the extraction ...of some structural parameters and the electron density profile of lipid bilayers. Here it is demonstrated that parametric modelling can be employed to determine the polydispersity of spherical or ellipsoidal vesicles and describe the electron density profile across the lipid bilayer. This approach is implemented in the computer program BILMIX. BILMIX delivers a description of the electron density of a lipid bilayer from SAXS data and simultaneously generates the corresponding size distribution of the unilamellar lipid vesicles.
The use of the BILMIX program to model small‐angle X‐ray scattering data of model membranes and deliver a description of the lipid bilayer electron density and vesicle size distribution is presented.
The trimeric periplasmic holdase chaperone Skp binds and stabilizes unfolded outer membrane proteins (OMPs) as part of bacterial OMP biogenesis. Skp binds client proteins in its central cavity, ...thereby reducing its backbone dynamics, but the molecular mechanisms that govern Skp dynamics and adaptation to differently sized clients remains unknown. Here, we employ a combination of microsecond timescale molecular dynamics simulation, small-angle X-ray scattering, and nuclear magnetic resonance spectroscopy to reveal that Skp is remarkably flexible, and features a molecular spring-loaded mechanism in its “tentacle” arms that enables switching between two distinct conformations on sub-millisecond timescales. The conformational switch is executed around a conserved pivot element within the coiled-coil structures of the tentacles, allowing expansion of the cavity and thus accommodation of differently sized clients. The spring-loaded mechanism shows how a chaperone can efficiently modulate its structure and function in an ATP-independent manner.
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•Skp chaperone binds diverse unfolded outer membrane protein substrates•Molecular basis for Skp promiscuity investigated via simulation, SAXS, and NMR•“Spring-loaded” dynamics enable switching between closed and open states•Energy-independent mechanism for efficient substrate capture over fast timescales
Holdbrook et al. combine simulations with spectroscopy to resolve a molecular “spring-loaded” mechanism in the flexible arms of a bacterial chaperone, helping to explain its substrate-binding promiscuity. The results demonstrate in atomic detail how a chaperone may efficiently modulate its structure in an ATP-independent manner.
PGC-1α is a crucial regulator of cellular metabolism and energy homeostasis that functionally acts together with the estrogen-related receptors (ERRα and ERRγ) in the regulation of mitochondrial and ...metabolic gene networks. Dimerization of the ERRs is a pre-requisite for interactions with PGC-1α and other coactivators, eventually leading to transactivation. It was suggested recently (Devarakonda et al) that PGC-1α binds in a strikingly different manner to ERRγ ligand-binding domains (LBDs) compared to its mode of binding to ERRα and other nuclear receptors (NRs), where it interacts directly with the two ERRγ homodimer subunits.
Here, we show that PGC-1α receptor interacting domain (RID) binds in an almost identical manner to ERRα and ERRγ homodimers. Microscale thermophoresis demonstrated that the interactions between PGC-1α RID and ERR LBDs involve a single receptor subunit through high-affinity, ERR-specific L3 and low-affinity L2 interactions. NMR studies further defined the limits of PGC-1α RID that interacts with ERRs. Consistent with these findings, the solution structures of PGC-1α/ERRα LBDs and PGC-1α/ERRγ LBDs complexes share an identical architecture with an asymmetric binding of PGC-1α to homodimeric ERR.
These studies provide the molecular determinants for the specificity of interactions between PGC-1α and the ERRs, whereby negative cooperativity prevails in the binding of the coactivators to these receptors. Our work indicates that allosteric regulation may be a general mechanism controlling the binding of the coactivators to homodimers.
The polypeptide growth factor, hepatocyte growth factor/scatter factor (HGF/SF), shares the multidomain structure and proteolytic mechanism of activation of plasminogen and other complex serine ...proteinases. HGF/SF, however, has no enzymatic activity. Instead, it controls the growth, morphogenesis, or migration of epithelial, endothelial, and muscle progenitor cells through the receptor tyrosine kinase MET. Using small-angle x-ray scattering and cryoelectron microscopy, we show that conversion of pro(single-chain)HGF/SF into the active two-chain form is associated with a major structural transition from a compact, closed conformation to an elongated, open one. We also report the structure of a complex between two-chain HGF/SF and the MET ectodomain (MET928) with 1:1 stoichiometry in which the N-terminal and first kringle domain of HGF/SF contact the face of the seven-blade β-propeller domain of MET harboring the loops connecting the β-strands b-c and d-a, whereas the C-terminal serine proteinase homology domain binds the opposite "b" face. Finally, we describe a complex with 2:2 stoichiometry between two-chain HGF/SF and a truncated form of the MET ectodomain (MET567), which is assembled around the dimerization interface seen in the crystal structure of the NK1 fragment of HGF/SF and displays the features of a functional, signaling unit. The study shows how the proteolytic mechanism of activation of the complex proteinases has been adapted to cell signaling in vertebrate organisms, offers a description of monomeric and dimeric ligand-receptor complexes, and provides a foundation to the structural basis of HGF/SF-MET signaling.
A series of carbosilane dendrimers of the 4th, 6th, and 7th generations with a terminal trimethylsilylsiloxane layer was synthesized. Theoretical models of these dendrimers were developed, and ...equilibrium dendrimer conformations obtained via molecular dynamics simulations were in a good agreement with experimental small-angle X-ray scattering (SAXS) data demonstrating molecule monodispersity and an almost spherical shape. It was confirmed that the glass transition temperature is independent of the dendrimer generation, but is greatly affected by the chemical nature of the dendrimer terminal groups. A sharp increase in the zero-shear viscosity of dendrimer melts was found between the 5th and the 7th dendrimer generations, which was qualitatively identical to that previously reported for polycarbosilane dendrimers with butyl terminal groups. The viscoelastic properties of high-generation dendrimers seem to follow some general trends with an increase in the generation number, which are determined by the regular branching structure of dendrimers.
Human MICAL1 is a member of a recently discovered family of multidomain proteins that couple a FAD‐containing monooxygenase‐like domain to typical protein interaction domains. Growing evidence ...implicates the NADPH oxidase reaction catalyzed by the flavoprotein domain in generation of hydrogen peroxide as a second messenger in an increasing number of cell types and as a specific modulator of actin filaments stability. Several proteins of the Rab families of small GTPases are emerging as regulators of MICAL activity by binding to its C‐terminal helical domain presumably shifting the equilibrium from the free – auto‐inhibited – conformation to the active one. We here extend the characterization of the MICAL1–Rab8 interaction and show that indeed Rab8, in the active GTP‐bound state, stabilizes the active MICAL1 conformation causing a specific four‐fold increase of kcat of the NADPH oxidase reaction. Kinetic data and small‐angle X‐ray scattering (SAXS) measurements support the formation of a 1:1 complex between full‐length MICAL1 and Rab8 with an apparent dissociation constant of approximately 8 μM. This finding supports the hypothesis that Rab8 is a physiological regulator of MICAL1 activity and shows how the protein region preceding the C‐terminal Rab‐binding domain may mask one of the Rab‐binding sites detected with the isolated C‐terminal fragment. SAXS‐based modeling allowed us to propose the first model of the free full‐length MICAL1, which is consistent with an auto‐inhibited conformation in which the C‐terminal region prevents catalysis by interfering with the conformational changes that are predicted to occur during the catalytic cycle.
Glutamate decarboxylase (Gad) catalyzes glutamate to γ-aminobutyrate conversion. Plant Gad is a ∼340 kDa hexamer, involved in development and stress response, and regulated by pH and binding of Ca
...2+/calmodulin (CaM) to the C-terminal domain. We determined the crystal structure of
Arabidopsis thaliana Gad1 in its CaM-free state, obtained a low-resolution structure of the calmodulin-activated Gad complex by small-angle X-ray scattering and identified the crucial residues, in the C-terminal domain, for regulation by pH and CaM binding. CaM activates Gad1 in a unique way by relieving two C-terminal autoinhibition domains of adjacent active sites, forming a 393 kDa Gad1–CaM complex with an unusual 1:3 stoichiometry. The complex is loosely packed: thanks to the flexible linkers connecting the enzyme core with the six C-terminal regulatory domains, the CaM molecules retain considerable positional and orientational freedom with respect to Gad1. The complex thus represents a prototype for a novel CaM-target interaction mode. Thanks to its two levels of regulation, both targeting the C-terminal domain, Gad can respond flexibly to different kinds of cellular stress occurring at different pH values.
Glutaminase C is a key metabolic enzyme, which is unregulated in many cancer systems and believed to play a central role in the Warburg effect, whereby cancer cells undergo changes to an altered ...metabolic profile. A long-standing hypothesis links enzymatic activity to the protein oligomeric state, hence the study of the solution behavior in general and the oligomer state in particular of glutaminase C is important for the understanding of the mechanism of protein activation and inhibition. In this report, this is extensively investigated in correlation to enzyme concentration or phosphate level, using a high-throughput microfluidic-mixing chip for the SAXS data collection, and we confirm that the oligomeric state correlates with activity. The in-depth solution behavior analysis further reveals the structural behavior of flexible regions of the protein in the dimeric, tetrameric and octameric state and investigates the C-terminal influence on the enzyme solution behavior. Our data enable SAXS-based rigid body modeling of the full-length tetramer states, thereby presenting the first ever experimentally derived structural model of mitochondrial glutaminase C including the N- and C-termini of the enzyme.
Structure and function of bacterial nucleoid is controlled by the nucleoid-associated proteins (NAP). In any phase of growth, various NAPs, acting sequentially, condense nucleoid and facilitate ...formation of its transcriptionally active structure. However, in the late stationary phase, only one of the NAPs, Dps protein, is strongly expressed, and DNA–protein crystals are formed that transform nucleoid into a static, transcriptionally inactive structure, effectively protected from the external influences. Discovery of crystal structures in living cells and association of this phenomenon with the bacterial resistance to antibiotics has aroused great interest in studying this phenomenon. The aim of this work is to obtain and compare structures of two related NAPs (HU and IHF), since they are the ones that accumulate in the cell at the late stationary stage of growth, which precedes formation of the protective DNA–Dps crystalline complex. For structural studies, two complementary methods were used in the work: small-angle X-ray scattering (SAXS) as the main method for studying structure of proteins in solution, and dynamic light scattering as a complementary one. To interpret the SAXS data, various approaches and computer programs were used (in particular, the evaluation of structural invariants, rigid body modeling and equilibrium mixture analysis in terms of the volume fractions of its components were applied), which made it possible to determine macromolecular characteristics and obtain reliable 3D structural models of various oligomeric forms of HU and IHF proteins with ~2 nm resolution typical for SAXS. It was shown that these proteins oligomerize in solution to varying degrees, and IHF is characterized by the presence of large oligomers consisting of initial dimers arranged in a chain. An analysis of the experimental and published data made it possible to hypothesize that just before the Dps expression, it is IHF that forms toroidal structures previously observed
in vivo
and prepares the platform for formation of DNA–Dps crystals. The results obtained are necessary for further investigation of the phenomenon of biocrystal formation in bacterial cells and finding ways to overcome resistance of various pathogens to external conditions.
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