Structural characterization of the active and inactive states of Src kinase in solution by small-angle X-ray scattering

Src kinase plays an important role in several signaling and regulation mechanisms in vivo. Enzymatic activity is tightly regulated through the phosphorylation and dephosphorylation of tyrosine 527, which is placed at the C-terminal tail. Here, we have addressed domain rearrangements involved in the regulation mechanism of Src kinase in solution using small-angle X-ray scattering. In the phosphorylated wild-type form of Src kinase corresponding to the inactive state of the protein, a single conformation compatible with a closed crystallographic structure was found in solution. In the Y527F point mutant representing the active state, analysis of scattering data reveals an equilibrium between two differently populated conformations differing in the radius of gyration by 5 Å. The major species (85% of the total population) presents a closed conformation indistinguishable from the crystallographic structure of the inactive state. The minor species (15% of the total population) is an open conformation similar to the crystallographic structure in the active state. The latter structure has the SH3, SH2, and SH2-catalytic domain linker assembled as a pseudo-two-domain protein. The regulation model emerging from this study, including at least three different conformational states, allows the tight regulation of the enzyme without compromising fast response in the presence of natural targets.     

Hinge stiffness is a barrier to RNA folding

Cation-mediated RNA folding from extended to compact, biologically active conformations relies on a temporal balance of forces. The Mg^2 +-mediated folding of the Tetrahymena thermophila ribozyme is characterized by rapid nonspecific collapse followed by tertiary-contact-induced compaction. This article focuses on an autonomously folding portion of the Tetrahymena ribozyme, its P4-P6 domain, in order to probe one facet of the rapid collapse: chain flexibility. The time evolution of P4-P6 folding was followed by global and local measures as a function of Mg^2 + concentration. While all concentrations of Mg^2 + studied are sufficient to screen the charge on the helices, the rates of compaction and tertiary contact formation diverge as the concentration of Mg^2 + increases; collapse is greatly accelerated by Mg^2 +, while tertiary contact formation is not. These studies highlight the importance of chain stiffness to RNA folding; at 10 mM Mg^2 +, a stiff hinge limits the rate of P4-P6 folding. At higher magnesium concentrations, the rate-limiting step shifts from hinge bending to tertiary contact formation.     

Changes in the molecular packing of fibrillin microfibrils during extension indicate intrafibrillar and interfibrillar reorganization in elastic response

Fibrillin-rich microfibrils are the major structural components of the extracellular matrix that provide elasticity in a majority of connective tissues. The basis of elastic properties lies in the organization of fibrillin molecules, which, unfortunately, is still poorly understood. An X-ray diffraction study of hydrated fibrillin-rich microfibrils from zonular filaments has been conducted to give an insight into the molecular structure of microfibrils in intact tissue. A series of measurements was taken during controlled tissue extension to observe alterations in the lateral packing of microfibrils. Computer-generated simulated patterns were used to fit the experimental X-ray scattering data and to obtain the fibril diameter and lateral distance between the fibrils. The results suggest a nonlinear correlation between external strain and decrease in fibril diameter and lateral spacing. This was accompanied by a nonlinear increase in axial periodicity and a structure with a 160-nm periodicity, which is reported here for the first time using X-ray diffraction. These changes may reflect the unraveling of fibrillin from the complex folded arrangement into a linear structure. This finding supports a pleating model where fibrillin molecules are highly folded within the microfibrils; more importantly, the connection is made between the interaction of individual microfibrils and the change in their suprafibrillar coherent organization during extension. We suggest that the intermediate states observed in our study reflect sequential unfolding of fibrillin and can explain the process of its reversible unraveling.     

Structural Characterization of Protein-Protein Complexes by Integrating Computational Docking with Small-angle Scattering Data

X-ray crystallography and NMR can provide detailed structural information of protein-protein complexes, but technical problems make their application challenging in the high-throughput regime. Other methods such as small-angle X-ray scattering (SAXS) are more promising for large-scale application, but at the cost of lower resolution, which is a problem that can be solved by complementing SAXS data with theoretical simulations. Here, we propose a novel strategy that combines SAXS data and accurate protein-protein docking simulations. The approach has been benchmarked on a large pool of known structures with synthetic SAXS data, and on three experimental examples. The combined approach (pyDockSAXS) provided a significantly better success rate (43% for the top 10 predictions) than either of the two methods alone. Further analysis of the influence of different docking parameters made it possible to increase the success rates for specific cases, and to define guidelines for improving the data-driven protein-protein docking protocols.     

The Ligand-Free State of the TPP Riboswitch: A Partially Folded RNA Structure

Riboswitches are elements of mRNA that regulate gene expression by undergoing structural changes upon binding of small ligands. Although the structures of several riboswitches have been solved with their ligands bound, the ligand-free states of only a few riboswitches have been characterized. The ligand-free state is as important for the functionality of the riboswitch as the ligand-bound form, but the ligand-free state is often a partially folded structure of the RNA, with conformational heterogeneity that makes it particularly challenging to study. Here, we present models of the ligand-free state of a thiamine pyrophosphate riboswitch that are derived from a combination of complementary experimental and computational modeling approaches. We obtain a global picture of the molecule using small-angle X-ray scattering data and use an RNA structure modeling software, MC-Sym, to fit local structural details to these data on an atomic scale. We have used two different approaches to obtaining these models. Our first approach develops a model of the RNA from the structures of its constituent junction fragments in isolation. The second approach treats the RNA as a single entity, without bias from the structure of its individual constituents. We find that both approaches give similar models for the ligand-free form, but the ligand-bound models differ for the two approaches, and only the models from the second approach agree with the ligand-bound structure known previously from X-ray crystallography. Our models provide a picture of the conformational changes that may occur in the riboswitch upon binding of its ligand. Our results also demonstrate the power of combining experimental small-angle X-ray scattering data with theoretical structure prediction tools in the determination of RNA structures beyond riboswitches.     

Structural Rearrangements Linked to Global Folding Pathways of the Azoarcus Group I Ribozyme

Stable RNAs must fold into specific three-dimensional structures to be biologically active, yet many RNAs form metastable structures that compete with the native state. Our previous time-resolved footprinting experiments showed that Azoarcus group I ribozyme forms its tertiary structure rapidly (τ < 30 ms) without becoming significantly trapped in kinetic intermediates. Here, we use stopped-flow fluorescence spectroscopy to probe the global folding kinetics of a ribozyme containing 2-aminopurine in the loop of P9. The modified ribozyme was catalytically active and exhibited two equilibrium folding transitions centered at 0.3 and 1.6 mM Mg²⁺, consistent with previous results. Stopped-flow fluorescence revealed four kinetic folding transitions with observed rate constants of 100, 34, 1, and 0.1 s^− 1 at 37 °C. From comparison with time-resolved Fe(II)-ethylenediaminetetraacetic acid footprinting of the modified ribozyme under the same conditions, these folding transitions were assigned to formation of the I_C intermediate, tertiary folding and docking of the nicked P9 tetraloop, reorganization of the P3 pseudoknot, and refolding of nonnative conformers, respectively. The footprinting results show that 50-60% of the modified ribozyme folds in less than 30 ms, while the rest of the RNA population undergoes slow structural rearrangements that control the global folding rate. The results show how small perturbations to the structure of the RNA, such as a nick in P9, populate kinetic folding intermediates that are not observed in the natural ribozyme.     

Crystal structure of a c-di-AMP riboswitch reveals an internally pseudo-dimeric RNA

Cyclic diadenosine monophosphate (c-di-AMP) is a second messenger that is essential for growth and homeostasis in bacteria. A recently discovered c-di-AMP-responsive riboswitch controls the expression of genes in a variety of bacteria, including important pathogens. To elucidate the molecular basis for specific binding of c-di-AMP by a gene-regulatory mRNA domain, we have determined the co-crystal structure of this riboswitch. Unexpectedly, the structure reveals an internally pseudo-symmetric RNA in which two similar three-helix-junction elements associate head-to-tail, creating a trough that cradles two c-di-AMP molecules making quasi-equivalent contacts with the riboswitch. The riboswitch selectively binds c-di-AMP and discriminates exquisitely against other cyclic dinucleotides, such as c-di-GMP and cyclic-AMP-GMP, via interactions with both the backbone and bases of its cognate second messenger. Small-angle X-ray scattering experiments indicate that global folding of the riboswitch is induced by the two bound cyclic dinucleotides, which bridge the two symmetric three-helix domains. This structural reorganization likely couples c-di-AMP binding to gene expression.     

The evidence of large-scale DNA-induced compaction in the mycobacterial chromosomal ParB

The bacterial chromosome trafficking apparatus or the segrosome participates in the mitotic-like segregation of the chromosomes prior to cell division in several bacteria. ParB, which is the parS DNA-binding component of the segrosome, polymerizes on the parS-adjacent chromosome to form a nucleoprotein filament of unknown nature for the segregation function. We combined static light scattering, circular dichroism and small-angle X-ray scattering to present evidence that the apo form of the mycobacterial ParB forms an elongated dimer with intrinsically disordered regions as well as folded domains in solution. A comparison of the solution scattering of the apo and the parS-bound ParBs indicates a rather drastic compaction of the protein upon DNA binding. We propose that this binding-induced conformational transition is priming the ParB for polymerization on the DNA template.     

Dissection of the ATP-Dependent Conformational Change Cycle of a Group II Chaperonin

Group II chaperonin captures an unfolded protein while in its open conformation and then mediates the folding of the protein during ATP-driven conformational change cycle. In this study, we performed kinetic analyses of the group II chaperonin from a hyperthermophilic archaeon, Thermococcus sp. KS-1 (TKS1-Cpn), by stopped-flow fluorometry and stopped-flow small-angle X-ray scattering to reveal the reaction cycle. Two TKS1-Cpn variants containing a Trp residue at position 265 or position 56 exhibit nearly the same fluorescence kinetics induced by rapid mixing with ATP. Fluorescence started to increase immediately after the start of mixing and reached a maximum at 1–2 s after mixing. Only in the presence of K⁺ that a gradual decrease in fluorescence was observed after the initial peak. Similar results were obtained by stopped-flow small-angle X-ray scattering. A rapid fluorescence increase, which reflects nucleotide binding, was observed for the mutant containing a Trp residue near the ATP binding site (K485W), irrespective of the presence or absence of K⁺. Without K⁺, a small, rapid fluorescence decrease followed the initial increase, and then a gradual decrease was observed. In contrast, with K⁺, a large, rapid fluorescence decrease occurred just after the initial increase, and then the fluorescence gradually increased. Finally, we observed ATP binding signal and also subtle conformational change in an ATPase-deficient mutant with K485W mutation. Based on these results, we propose a reaction cycle model for group II chaperonins.     

Evolution of quaternary structure in a homotetrameric enzyme

Dihydrodipicolinate synthase (DHDPS) is an essential enzyme in (S)-lysine biosynthesis and an important antibiotic target. All X-ray crystal structures solved to date reveal a homotetrameric enzyme. In order to explore the role of this quaternary structure, dimeric variants of Escherichia coli DHDPS were engineered and their properties were compared to those of the wild-type tetrameric form. X-ray crystallography reveals that the active site is not disturbed when the quaternary structure is disrupted. However, the activity of the dimeric enzymes in solution is substantially reduced, and a tetrahedral adduct of a substrate analogue is observed to be trapped at the active site in the crystal form. Remarkably, heating the dimeric enzymes increases activity. We propose that the homotetrameric structure of DHDPS reduces dynamic fluctuations present in the dimeric forms and increases specificity for the first substrate, pyruvate. By restricting motion in a key catalytic motif, a competing, non-productive reaction with a substrate analogue is avoided. Small-angle X-ray scattering and mutagenesis data, together with a B-factor analysis of the crystal structures, support this hypothesis and lead to the suggestion that in at least some cases, the evolution of quaternary enzyme structures might serve to optimise the dynamic properties of the protein subunits.     

Molecular architecture and structural transitions of a Clostridium thermocellum mini-cellulosome

The cellulosome is a highly elaborate cell-bound multienzyme complex that efficiently orchestrates the deconstruction of cellulose and hemicellulose, two of the nature's most abundant polymers. Understanding the intricacy of these nanomachines evolved by anaerobic microbes could sustain the development of an effective process for the conversion of lignocellulosic biomass to bio-ethanol. In Clostridium thermocellum, cellulosome assembly is mediated by high-affinity protein:protein interactions (> 10⁹ M^− 1) between dockerin modules found in the catalytic subunits and cohesin modules located in a non-catalytic protein scaffold termed CipA. Whereas the atomic structures of several cellulosomal components have been elucidated, the structural organization of the complete cellulosome remains elusive. Here, we reveal that a large fragment of the cellulosome presents a mostly compact conformation in solution, by solving the three-dimensional structure of a C. thermocellum mini-cellulosome comprising three consecutive cohesin modules, each bound to one Cel8A cellulase, at 35 Å resolution by cryo-electron microscopy. Interestingly, the three cellulosomal catalytic domains are found alternately projected outward from the CipA scaffold in opposite directions, in an arrangement that could expand the area of the substrate accessible to the catalytic domains. In addition, the cellulosome can transit from this compact conformation to a multitude of diverse and flexible structures, where the linkers between cohesin modules are extended and flexible. Thus, structural transitions controlled by changes in the degree of flexibility of linkers connecting consecutive cohesin modules could regulate the efficiency of substrate recognition and hydrolysis.     

The asymmetric ATPase cycle of the thermosome: elucidation of the binding, hydrolysis and product-release steps

Using a combination of intrinsic fluorescence to report ATP-induced rearrangements, quenched-flow to measure ATP hydrolysis "on-enzyme" and optical methods to probe the kinetics of product release, we have begun to dissect the process of energy transduction in the thermosome, a type II chaperonin from Thermoplasma acidophilum. Stoichiometric measurements of ATP binding reveal the tight association of eight nucleotide molecules per hexa-decamer, implying the filling of only one ring owing to strong negative cooperativity. After binding, we show that these eight ATP molecules are hydrolysed over the next 50 s, after which hydrolysis slows down markedly during the establishment of the steady state in the ATPase reaction, demonstrating that the kinetic system is off-rate limited. Looking in more detail, this rapid first-turnover can be dissected into two phases; the first occurring with a half-time of 0.8 s, the second with a half-time of 14 s, possibly reflecting the differential behaviour of the four alpha and four beta subunits in a single thermosome ring. To investigate the post-hydrolytic events, we used two heat-stable enzyme-linked optical assays to measure the rate of evolution of ADP and of phosphate from the thermosome active site. Neither product showed a rapid dissociation phase prior to the establishment of the steady state, showing that both are released slowly at a rate that limits the cycle. These data highlight the importance of the highly populated thermosome/ADP/Pi complex in the molecular mechanism.     

Structural Basis for Bivalent Smac-Mimetics Recognition in the IAP Protein Family

XIAP is an apoptotic regulator protein that binds to the effector caspases -3 and -7 through its BIR2 domain, and to initiator caspase-9 through its BIR3 domain. Molecular docking studies suggested that Smac-DIABLO may antagonize XIAP by concurrently targeting both BIR2 and BIR3 domains; on this basis bivalent Smac-mimetic compounds have been proposed and characterized. Here, we report the X-ray crystal structure of XIAP-BIR3 domain in complex with a two-headed compound (compound 3) with improved efficacy relative to its monomeric form. A small-angle X-ray scattering study of XIAP-BIR2BIR3, together with fluorescence polarization binding assays and compound 3 cytotoxicity tests on HL60 leukemia cell line are also reported. The crystal structure analysis reveals a network of interactions supporting XIAP-BIR3/compound 3 recognition; moreover, analytical gel-filtration chromatography shows that compound 3 forms a 1:1 stoichiometric complex with a XIAP protein construct containing both BIR2 and BIR3 domains. On the basis of the crystal structure and small-angle X-ray scattering, a model of the same BIR2-BIR3 construct bound to compound 3 is proposed, shedding light on the ability of compound 3 to relieve XIAP inhibitory effects on caspase-9 as well as caspases -3 and -7. A molecular modeling/docking analysis of compound 3 bound to cIAP1-BIR3 domain is presented, considering that Smac-mimetics have been shown to kill tumor cells by inducing cIAP1 and cIAP2 ubiquitination and degradation. Taken together, the results reported here provide a rationale for further development of compound 3 as a lead in the design of dimeric Smac mimetics for cancer treatment.     

Oligomerization propensity and flexibility of yeast frataxin studied by X-ray crystallography and small-angle X-ray scattering

Frataxin is a mitochondrial protein with a central role in iron homeostasis. Defects in frataxin function lead to Friedreich's ataxia, a progressive neurodegenerative disease with childhood onset. The function of frataxin has been shown to be closely associated with its ability to form oligomeric species; however, the factors controlling oligomerization and the types of oligomers present in solution are a matter of debate. Using small-angle X-ray scattering, we found that Co²⁺, glycerol, and a single amino acid substitution at the N-terminus, Y73A, facilitate oligomerization of yeast frataxin, resulting in a dynamic equilibrium between monomers, dimers, trimers, hexamers, and higher-order oligomers. Using X-ray crystallography, we found that Co²⁺ binds inside the channel at the 3-fold axis of the trimer, which suggests that the metal has an oligomer-stabilizing role. The results reveal the types of oligomers present in solution and support our earlier suggestions that the trimer is the main building block of yeast frataxin oligomers. They also indicate that different mechanisms may control oligomer stability and oligomerization in vivo.     

Activation of the zymogen to urokinase-type plasminogen activator is associated with increased interdomain flexibility

A key regulatory step for serine proteases of the trypsin clan is activation of the initially secreted zymogens, leading to an increase in activity by orders of magnitude. Zymogen activation occurs by cleavage of a single peptide bond near the N-terminus of the catalytic domain. Besides the catalytic domain, most serine proteases have N-terminal A-chains with independently folded domains. Little is known about how zymogen activation affects the interplay between domains. This question is investigated with urokinase-type plasminogen activator (uPA), which has an epidermal growth factor domain and a kringle domain, connected to the catalytic domain by a 15-residue linker. uPA has been implicated under several pathological conditions, and one possibility for pharmacological control is targeting the conversion of the zymogen pro-uPA to active uPA. Therefore, a small-angle X-ray scattering study of the conformations of pro-uPA and uPA in solution was performed. Structural models for the proteins were derived using available atomic-resolution structures for the various domains. Active uPA was found to be flexible with a random conformation of the amino-terminal fragment domain with respect to the serine protease domain. In contrast, pro-uPA was observed to be rigid, with the amino-terminal fragment domain in a fixed position with respect to the serine protease domain. Analytical ultracentrifugation analysis supported the observed difference between pro-uPA and uPA in overall shape and size seen with small-angle X-ray scattering. Upon association of either of two monoclonal Fab (fragment antigen-binding) fragments that are directed against the catalytic domain of, respectively, pro-uPA and uPA, rigid structures were formed.     

A Method for Helical RNA Global Structure Determination in Solution Using Small-Angle X-Ray Scattering and NMR Measurements

We report a “top-down” method that uses mainly duplexes' global orientations and overall molecular dimension and shape restraints, which were extracted from experimental NMR and small-angle X-ray scattering data, respectively, to determine global architectures of RNA molecules consisting of mostly A-form-like duplexes. The method is implemented in the G2G (from global measurement to global structure) toolkit of programs. We demonstrate the efficiency and accuracy of the method by determining the global structure of a 71-nt RNA using experimental data. The backbone root-mean-square deviation of the ensemble of the calculated global structures relative to the X-ray crystal structure is 3.0 ± 0.3 Å using the experimental data and is only 2.5 ± 0.2 Å for the three duplexes that were orientation restrained during the calculation. The global structure simplifies interpretation of multidimensional nuclear Overhauser spectra for high-resolution structure determination. The potential general application of the method for RNA structure determination is discussed.     

Structure and thermodynamics of transient protein-protein complexes by chemometric decomposition of SAXS datasets

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Shape and subunit organisation of the DNA methyltransferase M.AhdI by small-angle neutron scattering

Type I restriction-modification (R-M) systems encode multisubunit/multidomain enzymes. Two genes (M and S) are required to form the methyltransferase (MTase) that methylates a specific base within the recognition sequence and protects DNA from cleavage by the endonuclease. The DNA methyltransferase M.AhdI is a 170 kDa tetramer with the stoichiometry M(2)S(2) and has properties typical of a type I MTase. The M.AhdI enzyme has been prepared with deuterated S subunits, to allow contrast variation using small-angle neutron scattering (SANS) methods. The SANS data were collected in a number of (1)H:(2)H solvent contrasts to allow matching of one or other of the subunits in the multisubunit enzyme. The radius of gyration (R(g)) and maximum dimensions (D(max)) of the M subunits in situ in the multisubunit enzyme (50 A and 190 A, respectively) are close of those of the entire MTase (51 A and 190 A). In contrast, the S subunits in situ have experimentally determined values of R(g)=35 A and D(max)=110 A, indicating their more central location in the enzyme. Ab initio reconstruction methods yield a low-resolution structural model of the shape and subunit organization of M.AhdI, in which the Z-shaped structure of the S subunit dimer can be discerned. In contrast, the M subunits form a much more elongated and extended structure. The core of the MTase comprises the two S subunits and the globular regions of the two M subunits, with the extended portion of the M subunits most probably forming highly mobile regions at the outer extremities, which collapse around the DNA when the MTase binds.     

X-ray and neutron small-angle scattering analysis of the complex formed by the Met receptor and the Listeria monocytogenes invasion protein InlB

The Listeria monocytogenes surface protein InlB binds to the extracellular domain of the human receptor tyrosine kinase Met, the product of the c-met proto-oncogene. InlB binding activates the Met receptor, leading to uptake of Listeria into normally nonphagocytic host cells. The N-terminal half of InlB (InlB₃₂₁) is sufficient for Met binding and activation. The complex between this Met-binding domain of InlB and various constructs of the Met ectodomain was characterized by size exclusion chromatography and dynamic light scattering, and structural models were built using small-angle X-ray scattering and small-angle neutron scattering. Although most receptor tyrosine kinase ligands induce receptor dimerization, InlB₃₂₁ consistently binds the Met ectodomain with a 1:1 stoichiometry. A construct comprising the Sema and PSI domains of Met, although sufficient to bind the physiological Met ligand hepatocyte growth factor/scatter factor, does not form a complex with InlB₃₂₁ in solution, highlighting the importance of Met Ig domains for InlB binding. Small-angle X-ray scattering and small-angle neutron scattering measurements of ligand and receptor, both free and in complex, reveal an elongated shape for the receptor. The four Ig domains form a bent, rather than a fully extended, conformation, and InlB₃₂₁ binds to Sema and the first Ig domain of Met, in agreement with the recent crystal structure of a smaller Met fragment in complex with InlB₃₂₁. These results call into question whether receptor dimerization is the basic underlying event in InlB₃₂₁-mediated Met activation and demonstrate differences in the mechanisms by which the physiological ligand hepatocyte growth factor/scatter factor and InlB₃₂₁ bind and activate the Met receptor.