The quaternary structure of Escherichia coli N-acetylneuraminate lyase is essential for functional expression

As part of a general study into the impact of quaternary structure on enzyme function, a library of 31 point mutations were engineered at the dimer-dimer interface of the homotetrameric (β/α)₈-barrel protein, N-acetylneuraminate lyase (NAL, EC 4.1.3.3). Disruption of the interface generated either soluble tetramers or putative dimers that were absolutely insoluble and inactive. Intriguingly, the soluble tetramers were found to have widely varying k_cat values, hinting at a role for the interface in catalysis. Leucine 171 was identified as essential to interface integrity. We conclude that the dimer-dimer interface of NAL is intolerant to mutation and essential for functional expression.     

Conserved central domains control the quaternary structure of type I and type II Hsp40 molecular chaperones

Heat shock protein (Hsp)40s play an essential role in protein metabolism by regulating the polypeptide binding and release cycle of Hsp70. The Hsp40 family is large, and specialized family members direct Hsp70 to perform highly specific tasks. Type I and Type II Hsp40s, such as yeast Ydj1 and Sis1, are homodimers that dictate functions of cytosolic Hsp70, but how they do so is unclear. Type I Hsp40s contain a conserved, centrally located cysteine-rich domain that is replaced by a glycine- and methionine-rich region in Type II Hsp40s, but the mechanism by which these unique domains influence Hsp40 structure and function is unknown. This is the case because high-resolution structures of full-length forms of these Hsp40s have not been solved. To fill this void, we built low-resolution models of the quaternary structure of Ydj1 and Sis1 with information obtained from biophysical measurements of protein shape, small-angle X-ray scattering, and ab initio protein modeling. Low-resolution models were also calculated for the chimeric Hsp40s YSY and SYS, in which the central domains of Ydj1 and Sis1 were exchanged. Similar to their human homologs, Ydj1 and Sis1 each has a unique shape with major structural differences apparently being the orientation of the J domains relative to the long axis of the dimers. Central domain swapping in YSY and SYS correlates with the switched ability of YSY and SYS to perform unique functions of Sis1 and Ydj1, respectively. Models for the mechanism by which the conserved cysteine-rich domain and glycine- and methionine-rich region confer structural and functional specificity to Type I and Type II Hsp40s are discussed.     

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.     

Structural transitions and thermodynamics of a glycine-dependent riboswitch from Vibrio cholerae

Riboswitches are complex folded RNA domains found in noncoding regions of mRNA that regulate gene expression upon small molecule binding. Recently, Breaker and coworkers reported a tandem aptamer riboswitch (VCI-II) that binds glycine cooperatively. Here, we use hydroxyl radical footprinting and small-angle X-ray scattering (SAXS) to study the conformations of this tandem aptamer as a function of Mg(2+) and glycine concentration. We fit a simple three-state thermodynamic model that describes the energetic coupling between magnesium-induced folding and glycine binding. Furthermore, we characterize the structural conformations of each of the three states: In low salt with no magnesium present, the VCI-II construct has an extended overall conformation, presumably representing unfolded structures. Addition of millimolar concentrations of Mg(2+) in the absence of glycine leads to a significant compaction and partial folding as judged by hydroxyl radical protections. In the presence of millimolar Mg(2+) concentrations, the tandem aptamer binds glycine cooperatively. The glycine binding transition involves a further compaction, additional tertiary packing interactions and further uptake of magnesium ions relative to the state in high Mg(2+) but no glycine. Employing density reconstruction algorithms, we obtain low resolution 3-D structures for all three states from the SAXS measurements. These data provide a first glimpse into the structural conformations of the VCI-II aptamer, establish rigorous constraints for further modeling, and provide a framework for future mechanistic studies.     

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.     

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.     

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.     

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.     

Resistance of α-crystallin quaternary structure to UV irradiation

The damaging effect of UV radiation (λ > 260 nm) on bovine α-crystallin in solution was studied by small-angle X-ray scattering, gel permeation chromatography, electrophoresis, absorption and fluorescence spectroscopy, and differential scanning calorimetry. The results obtained show that damage to even a large number of subunits within an α-crystallin oligomer does not cause significant rearrangement of its quaternary structure, aggregation of oligomers, or the loss of their solubility. Due to the high resistance of its quaternary structure, α-crystallin is able to prevent aggregation of destabilized proteins (especially of γ- and β-crystallins) and so to maintain lens transparency throughout the life of an animal (the chaperone-like function of α-crystallin).     

Crystal structure of human filamin C domain 23 and small angle scattering model for filamin C 23-24 dimer

Filamin C is a dimeric, actin-binding protein involved in organization of cortical cytoskeleton and of the sarcomere. We performed crystallographic, small-angle X-ray scattering and analytical ultracentrifugation experiments on the constructs containing carboxy-terminal domains of the protein (domains 23-24 and 19-21). The crystal structure of domain 23 of filamin C showed that the protein adopts the expected immunoglobulin (Ig)-like fold. Small-angle X-ray scattering experiments performed on filamin C tandem Ig-like domains 23 and 24 reveal a dimer that is formed by domain 24 and that domain 23 has little interactions with itself or with domain 24, while the analytical ultracentrifugation experiments showed that the filamin C domains 19-21 form elongated monomers in diluted solutions.     

Fibrillogenesis in dense collagen solutions: a physicochemical study

Fibrillogenesis, the formation of collagen fibrils, is a key factor in connective tissue morphogenesis. To understand to what extent cells influence this process, we systematically studied the physicochemistry of the self-assembly of type I collagen molecules into fibrils in vitro. We report that fibrillogenesis in solutions of type I collagen, in a high concentration range close to that of living tissues (40-300 mg/ml), yields strong gels over wide pH and ionic strength ranges. Structures of gels were described by combining microscopic observations (transmission electron microscopy) with small- and wide-angle X-ray scattering analysis, and the influence of concentration, pH, and ionic strength on the fibril size and organization was evaluated. The typical cross-striated pattern and the corresponding small-angle X-ray scattering 67-nm diffraction peaks were visible in all conditions in the pH 6 to pH 12 range. In reference conditions (pH 7.4, ionic strength = 150 mM, 20 °C), collagen concentration greatly influences the overall macroscopic structure of the resultant fibrillar gels, as well as the morphology and structure of the fibrils themselves. At a given collagen concentration, increasing the ionic strength from 24 to 261 mM produces larger fibrils until the system becomes biphasic. We also show that fibrils can form in acidic medium (pH ∼ 2.5) at very high collagen concentrations, beyond 150 mg/ml, which suggests a possible cholesteric-to-smectic phase transition. This set of data demonstrates how simple physicochemical parameters determine the molecular organization of collagen. Such an in vitro model allows us to study the intricate process of fibrillogenesis in conditions of molecular packing close to that which occurs in biological tissue morphogenesis.     

Loosening of the phage structure in a low ionic strength environment

Structural parameters of phage T7 were compared in two frequently use Tris buffers of high and low ionic strength, in order to explain the different biological activity and drug-binding characteristics.Characteristics of the whole phage geometry were obtained by viscosimetry, static and quasi-elastic light-scattering and small-angle X-ray scattering. The latter method revealed dissimilarities in the intraphage DNA compactness, consistent with the findings of the optical absorption melting studies.Alterations in the particle dimensions determined in the same sample by different methods are discussed, and a model is constructed to explain the structural modifications that occur on lowering the ionic strength.     

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.     

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.     

Towards a molecular description of intermediate filament structure and assembly

Intermediate filaments (IFs) represent one of the prominent cytoskeletal elements of metazoan cells. Their constituent proteins are coded by a multigene family, whose members are expressed in complex patterns that are controlled by developmental programs of differentiation. Hence, IF proteins found in epidermis differ significantly from those in muscle or neuronal tissues. Due to their fibrous nature, which stems from a fairly conserved central alpha-helical coiled-coil rod domain, IF proteins have long resisted crystallization and thus determination of their atomic structure. Since they represent the primary structural elements that determine the shape of the nucleus and the cell more generally, a major challenge is to arrive at a more rational understanding of how their nanomechanical properties effect the stability and plasticity of cells and tissues. Here, we review recent structural results of the coiled-coil dimer, assembly intermediates and growing filaments that have been obtained by a hybrid methods approach involving a rigorous combination of X-ray crystallography, small angle X-ray scattering, cryo-electron tomography, computational analysis and molecular modeling.     

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.     

Characterization of protein fold by wide-angle X-ray solution scattering

Wide-angle X-ray solution scattering (WAXS) patterns contain substantial information about the three-dimensional structure of a protein. Although WAXS data have far less information than is required for determination of a full three-dimensional structure, the actual amount of information contained in a WAXS pattern has not been carefully quantified. Here we carry out an analysis of the amount of information that can be extracted from a WAXS pattern and demonstrate that it is adequate to estimate the secondary-structure content of a protein and to strongly limit its possible tertiary structures. WAXS patterns computed from the atomic coordinates of a set of 498 protein domains representing all of known fold space were used as the basis for constructing a multidimensional space of all corresponding WAXS patterns (‘WAXS space’). Within WAXS space, each scattering pattern is represented by a single vector. A principal components analysis was carried out to identify those directions in WAXS space that provide the greatest discrimination among patterns. The number of dimensions that provide significant discrimination among protein folds agrees well with the number of independent parameters estimated from a naïve Shannon sampling theorem approach. Estimates of the relative abundances of secondary structures were made using training/test sets derived from this data set. The average error in the estimate of α-helical content was 11%, and of β-sheet content was 9%. The distribution of proteins that are members of the four structure classes, α, β, α/β and α+β, are well separated in WAXS space when data extending to a spacing of 2.2 Å are used. Quantification of the information embedded within a WAXS pattern indicates that these data can be used as a powerful constraint in homology modeling of protein structures.     

Asparagine-84, a regulatory allosteric site residue,helps maintain the quaternary structure of Campylobacter jejuni dihydrodipicolinate synthase

Dihydrodipicolinate synthase (DHDPS) from Campylobacter jejuni is a natively homotetrameric enzyme that catalyzes the first unique reaction of (S)-lysine biosynthesis and is feedback-regulated by lysine through binding to an allosteric site. High-resolution structures of the DHDPS-lysine complex have revealed significant insights into the binding events. One key asparagine residue, N84, makes hydrogen bonds with both the carboxyl and the α-amino group of the bound lysine. We generated two mutants, N84A and N84D, to study the effects of these changes on the allosteric site properties. However, under normal assay conditions, N84A displayed notably lower catalytic activity, and N84D showed no activity. Here we show that these mutations disrupt the quaternary structure of DHDPS in a concentration-dependent fashion, as demonstrated by size-exclusion chromatography, multi-angle light scattering, dynamic light scattering, small-angle X-ray scattering (SAXS) and high-resolution protein crystallography.