Molecular structure and dynamics of the dimeric human small heat shock protein HSPB6

ATP-independent small heat-shock proteins (sHSPs) are an essential component of the cellular chaperoning machinery. Under both normal and stress conditions, sHSPs bind partially unfolded proteins and prevent their irreversible aggregation. Canonical vertebrate sHSPs, such as the α-crystallins, form large polydisperse oligomers from which smaller, functionally active subspecies dissociate. Here we focus on human HSPB6 which, despite having considerable homology to the α-crystallins in both the N-terminal region and the signature α-crystallin domain (ACD), only forms dimers in solution that represent the basic chaperoning subspecies. We addressed the three-dimensional structure and functional properties of HSPB6 in a hybrid study employing X-ray crystallography, solution small-angle X-ray scattering (SAXS), mutagenesis, size-exclusion chromatography and chaperoning assays. The crystal structure of a proteolytically stable fragment reveals typical ACD dimers which further form tetrameric assemblies as a result of extensive inter-dimer patching of the β4/β8 grooves. The patching is surprisingly mediated by tripeptide motifs, found in the N-terminal domain directly adjacent to the ACD, that are resembling but distinct from the canonical IxI sequence commonly binding this groove. By combining the crystal structure with SAXS data for the full-length protein, we derive a molecular model of the latter. In solution, HSPB6 shows a strong attractive self-interaction, a property that correlates with its chaperoning activity. Both properties are dictated by the unstructured yet compact N-terminal domain, specifically a region highly conserved across vertebrate sHSPs.     

Intramolecular interactions regulate SAP97 binding to GKAP

Membrane-associated guanylate kinase homologs (MAGUKs) are multidomain proteins found to be central organizers of cellular junctions. In this study, we examined the molecular mechanisms that regulate the interaction of the MAGUK SAP97 with its GUK domain binding partner GKAP (GUK-associated protein). The GKAP-GUK interaction is regulated by a series of intramolecular interactions. Specifically, the association of the Src homology 3 (SH3) domain and sequences situated between the SH3 and GUK domains with the GUK domain was found to interfere with GKAP binding. In contrast, N-terminal sequences that precede the first PDZ domain in SAP97, facilitated GKAP binding via its association with the SH3 domain. Utilizing crystal structure data available for PDZ, SH3 and GUK domains, molecular models of SAP97 were generated. These models revealed that SAP97 can exist in a compact U-shaped conformation in which the N-terminal domain folds back and interacts with the SH3 and GUK domains. These models support the biochemical data and provide new insights into how intramolecular interactions may regulate the association of SAP97 with its binding partners.     

A compact intermediate state of calmodulin in the process of target binding

Calmodulin undergoes characteristic conformational changes by binding Ca(2+), which allows it to bind to more than 300 target proteins and regulate numerous intracellular processes in all eukaryotic cells. We measured the conformational changes of calmodulin upon Ca(2+) and mastoparan binding using the time-resolved small-angle X-ray scattering technique combined with flash photolysis of caged calcium. This measurement system covers the time range of 0.5-180 ms. Within 10 ms of the stepwise increase in Ca(2+) concentration, we identified a distinct compact conformational state with a drastically different molecular dimension. This process is too fast to study with a conventional stopped-flow apparatus. The compact conformational state was also observed without mastoparan, indicating that the calmodulin forms a compact globular conformation by itself upon Ca(2+) binding. This new conformational state of calmodulin seems to regulate Ca(2+) binding and conformational changes in the N-terminal domain. On the basis of this finding, an allosteric mechanism, which may have implications in intracellular signal transduction, is proposed.     

Small-angle X-ray scattering and NMR studies of the conformation of the PDZ region of SAP97 and its interactions with Kir2.1

The functional localization of potassium inward rectifiers is regulated by SAP97, a PDZ membrane-associated guanylate kinase protein. We describe here an investigation of the conformation of the PDZ domain region of SAP97 PDZ1-3. The NMR and SAXS data reveal conformational dynamics. The NMR data show minimal interdomain contacts, with the U3 linker region between PDZ2 and PDZ3 being largely unstructured. Shape analysis of the SAXS profiles revealed a dumbbell for the PDZ12 double domain. An overall elongated, asymmetric shape comprised of two to three distinct components characterizes the triple domain PDZ1-3. In addition, rigid body modeling shows that the representative average shape does not provide the full picture and that the data for the triple domain are consistent with large variations, suggesting significant conformational flexibility. However, the dynamics appears to be restricted as PDZ3 is located essentially within approximately 40 A from PDZ12. We also show that the Kir2.1 cytoplasmic domain interacts with all three PDZ domains but with a clear preference for PDZ2 even in the presence of the U3 region. We speculate that the restricted dynamics and preferential Kir2.1 binding to PDZ2 are features that enable SAP97 to function as a scaffold protein, allowing other proteins each to bind to the other two PDZ domains in sufficient proximity to yield productive channelosomes.     

SAP97 increases Kv1.5 currents through an indirect N-terminal mechanism

The functional interaction of the voltage-gated potassium channel hKv1.5 with the PDZ domain containing protein SAP97 has been investigated. In marked contrast with the known dependence of SAP97-induced Kv1 potassium current down-regulation on the channel C-termini, SAP97 increased hKv1.5 current through an indirect interaction with the Kv1.5 N-terminus. Deletion of the Kv1.5 N-terminus eliminated the SAP97-mediated increase in potassium currents whereas deletion of the channel's C-terminal PDZ binding motif had no effect. In contrast with other Kv1-SAP97 interactions, no physical interaction could be detected in vivo or in vitro between the two proteins. The proteins did not co-localize in cardiac myocytes nor did they co-immunoprecipitate from transfected HEK cells. Yeast two-hybrid experiments also failed to detect any interaction between the two proteins, but in one experiment of six, Kv1.5 co-immunoprecipitated very inefficiently with SAP97 from rat ventricular myocytes. Thus, we conclude that the influence of SAP97 on Kv1.5 potassium current levels is dependent upon a novel regulatory mechanism.     

Disease-associated polyglutamine stretches in monomeric huntingtin adopt a compact structure

Abnormal polyglutamine (polyQ) tracts are the only common feature in nine proteins that each cause a dominant neurodegenerative disorder. In Huntington's disease, tracts longer than 36 glutamines in the protein huntingtin (htt) cause degeneration. In situ, monoclonal antibody 3B5H10 binds to different htt fragments in neurons in proportion to their toxicity. Here, we determined the structure of 3B5H10 Fab to 1.9?? resolution by X-ray crystallography. Modeling demonstrates that the paratope forms a groove suitable for binding two β-rich polyQ strands. Using small-angle X-ray scattering, we confirmed that the polyQ epitope recognized by 3B5H10 is a compact two-stranded hairpin within monomeric htt and is abundant in htt fragments unbound to antibody. Thus, disease-associated polyQ stretches preferentially adopt compact conformations. Since 3B5H10 binding predicts degeneration, this compact polyQ structure may be neurotoxic.     

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.     

Structural differences between the SH3-HOOK-GuK domains of SAP90/PSD-95 and SAP97

The SH3-HOOK-GUK domains of the postsynaptic scaffolding proteins SAP90/PSD-95 and SAP97 are established targets of synaptic plasticity processes in the brain. A crucial molecular mechanism involved is the transition of this domain to different conformational states. We purified the SH3-HOOK-GUK domain of both proteins to examine variations in protein conformation and stability. As monitored by circular dichroism and differential scanning calorimetry, SAP97 (T_m = 64 °C) is significantly more thermal stable than SAP90/PSD-95 (T_m = 52 °C) and follows a bimodal phase transition. GdmCl-induced equilibrium unfolding of both proteins follows the two-state transitions and thus does not involve the accumulation of stable intermediate state(s). Equilibrium unfolding of SAP97 is highly cooperative from a native state to an unfolded state. In contrast, SAP90/PSD-95 follows a non-cooperative transition from native to unfolded states. A highly cooperative unfolding reaction in case of SAP97 indicates that the protein existed initially as a compact, well-folded structure, while the gradual, non-cooperative melting reaction in case of SAP90/PSD-95 indicates that the protein is in comparison more flexible.     

Three-dimensional structure of α-crystallin domain dimers of human small heat shock proteins HSPB1 and HSPB6

Small heat shock proteins (sHSPs) are a family of evolutionary conserved ATP-independent chaperones. These proteins share a common architecture defined by a signature α-crystallin domain (ACD) flanked by highly variable N- and C-terminal extensions. The ACD, which has an immunoglobulin-like fold, plays an important role in sHSP assembly. This domain mediates dimer formation of individual protomers, which then may assemble into larger oligomers. In vertebrate sHSPs, the dimer interface is formed by the symmetrical antiparallel pairing of two β-strands (β7), generating an extended β-sheet on one face of the ACD dimer. Recent structural studies of isolated ACDs from a number of vertebrate sHSPs suggest a variability in the register of the β7/β7 strand interface, which may, in part, give rise to the polydispersity often associated with the full-length proteins. To further analyze the structure of ACD dimers, we have employed a combination of X-ray crystallography and solution small-angle X-ray scattering (SAXS) to study the ACD-containing fragments of human HSPB1 (HSP27) and HSPB6 (HSP20). Unexpectedly, the obtained crystal structure of the HSPB1 fragment does not reveal the typical β7/β7 dimers but, rather, hexamers formed by an asymmetric contact between the β4 and the β7 strands from adjacent ACDs. Nevertheless, in solution, both ACDs form stable dimers via the symmetric antiparallel interaction of β7 strands. Using SAXS, we show that it is possible to discriminate between different putative registers of the β7/β7 interface, with the results indicating that, under physiological conditions, there is only a single register of the strands for both proteins.     

Solution structure of the two-iron rubredoxin of Pseudomonas oleovorans determined by NMR spectroscopy and solution X-ray scattering and interactions with rubredoxin reductase

Here we provide insights into the molecular structure of the two-iron 19-kDa rubredoxin (AlkG) of Pseudomonas oleovorans using solution-state nuclear magnetic resonance (NMR) and small-angle X-ray scattering studies. Sequence alignment and biochemical studies have suggested that AlkG comprises two rubredoxin folds connected by a linker region of approximately 70 amino acid residues. The C-terminal domain (C-Rb) of this unusual rubredoxin, together with approximately 35 amino acid residues of the predicted linker region, was expressed in Escherichia coli, purified in the one-iron form and the structure of the cadmium-substituted form determined at high-resolution by NMR spectroscopy. The structure shows that the C-Rb domain is similar in fold to the conventional one-iron rubredoxins from other organisms, whereas the linker region does not have any discernible structure. This tandem "flexible-folded" structure of the polypeptide chain derived for the C-Rb protein was confirmed using solution X-ray scattering methods. X-ray scattering studies of AlkG indicated that the 70-amino acid residue linker forms a structured, yet mobile, polypeptide segment connecting the globular N- and C-terminal domains. The X-ray scattering studies also showed that the N-terminal domain (N-Rb) has a molecular conformation similar to that of C-Rb. The restored molecular shape indicates that the folded N-Rb and C-Rb domains of AlkG are noticeably separated, suggesting some domain movement on complex formation with rubredoxin reductase to allow interdomain electron transfer between the metal centers in AlkG. This study demonstrates the advantage of combining X-ray scattering and NMR methods in structural studies of dynamic, multidomain proteins that are not suited to crystallographic analysis. The study forms a structural foundation for functional studies of the interaction and electron-transfer reactions of AlkG with rubredoxin reductase, also reported herein.     

The shapes of biantennary and tri/tetraantennary alpha 1 acid glycoprotein by small-angle neutron and X-ray scattering

Two forms of alpha 1 acid glycoprotein (orosomucoid) have been studied using small-angle neutron and X-ray scattering techniques; in one form all the five glycan chains were biantennary, while in the other they were either triantennary or tetraantennary. The radius of gyration RG was found to be sensitive to salt for the biantennary form, but to be unchanged up to an ionic strength of 3 M for the triantennary and tetraantennary forms. Conformational heterogeneity is thus associated with carbohydrate heterogeneity. Hydrodynamic frictional coefficients confirm these findings. Simple models of alpha 1 acid glycoprotein were developed to account for the RG and values. These show that the compact conformation is slightly more elongated than a globular protein and that the expanded biantennary conformation has a most extended carbohydrate structure. Up to half of the surface of the compact shape can be covered by carbohydrate residues.     

Identification of multiple binding partners for the amino-terminal domain of synapse-associated protein 97

Multiprotein complexes mediate static and dynamic functions to establish and maintain cell polarity in both epithelial cells and neurons. Membrane-associated guanylate kinase (MAGUK) proteins are thought to be scaffolding molecules in these processes and bind multiple proteins via their obligate postsynaptic density (PSD)-95/Disc Large/Zona Occludens-1, Src homology 3, and guanylate kinase-like domains. Subsets of MAGUK proteins have additional protein-protein interaction domains. An additional domain we identified in SAP97 called the MAGUK recruitment (MRE) domain binds the LIN-2,7 amino-terminal (L27N) domain of mLIN-2/CASK, a MAGUK known to bind mLIN-7. Here we show that SAP97 binds two other mLIN-7 binding MAGUK proteins. One of these MAGUK proteins, DLG3, coimmunoprecipitates with SAP97 in lysates from rat brain and transfected Madin-Darby canine kidney cells. This interaction requires the MRE domain of SAP97 and surprisingly, both the L27N and L27 carboxyl-terminal (L27C) domains of DLG3. We also demonstrate that SAP97 can interact with the MAGUK protein, DLG2, but not the highly related protein, PALS2. The ability of SAP97 to interact with multiple MAGUK proteins is likely to be important for the targeting of specific protein complexes in polarized cells.     

The compact and expanded denatured conformations of apomyoglobin in the methanol-water solvent

We have performed a detailed study of methanol-induced conformational transitions of horse heart apomyoglobin (apoMb) to investigate the existence of the compact and expanded denatured states. A combination of far- and near-ultraviolet circular dichroism, NMR spectroscopy, and small-angle X-ray scattering (SAXS) was used, allowing a phase diagram to be constructed as a function of pH and the methanol concentration. The phase diagram contains four conformational states, the native (N), acid-denatured (U(A)), compact denatured (I(M)), and expanded helical denatured (H) states, and indicates that the compact denatured state (I(M)) is stable under relatively mild denaturing conditions, whereas the expanded denatured states (U(A) and H) are realized under extreme conditions of pH (strong electric repulsion) or alcohol concentration (weak hydrophobic interaction). The results of this study, together with many previous studies in the literature, indicate the general existence of the compact denatured states not only in the salt-pH plane but also in the alcohol-pH plane. Furthermore, to determine the general feature of the H conformation we used several proteins including ubiquitin, ribonuclease A, alpha-lactalbumin, beta-lactoglobulin, and Streptomyces subtilisin inhibitor (SSI) in addition to apoMb. SAXS studies of these proteins in 60% methanol showed that the H states of these all proteins have expanded and nonglobular conformations. The qualitative agreement of the experimental data with computer-simulated Kratky profiles also supports this structural feature of the H state.     

Structural characteristics of alpha-synuclein oligomers stabilized by the flavonoid baicalein

The flavonoid baicalein inhibits fibrillation of α-synuclein, which is a major component of Lewy bodies in Parkinson's disease. It has been known that baicalein induces the formation of α-synuclein oligomers and consequently prevents their fibrillation. In order to evaluate the structural properties of baicalein-stabilized oligomers, we purified oligomer species by HPLC and examined their stability and structure by CD, Fourier transform infrared spectroscopy, size exclusion chromatography HPLC, small-angle X-ray scattering, and atomic force microscopy. Baicalein-stabilized oligomers are β-sheet-enriched according to CD and Fourier transform infrared spectroscopy analyses. They did not form fibrils even after very prolonged incubation. From small-angle X-ray scattering data and atomic force microscopy images, the oligomers were characterized as quite compact globular species. Oligomers were extremely stable, with a GdmCl C_m = 3.3 M. This high stability explains the previously observed inhibition properties of baicalein against α-synuclein fibrillation. These baicalein-stabilized oligomers, added to the solution of aggregating α-synuclein, were able to noticeably inhibit its fibrillation. After prolonged coincubation, short fibrils were formed, suggesting an effective interaction of oligomers with monomeric α-synuclein. Membrane permeability tests suggested that the baicalein-stabilized oligomers had a mild effect on the integrity of the membrane surface. This effect was rather similar to that of the monomeric protein, suggesting that targeted stabilization of certain α-synuclein oligomers might offer a potential strategy for the development of novel Parkinson's disease therapies.     

Quaternary structure, protein dynamics, and synaptic function of SAP97 controlled by L27 domain interactions

Single-particle electron microscopy (EM) combined with biochemical measurements revealed the molecular shape of SAP97 and a monomer-dimer transition that depended on the N-terminal L27 domain. Overexpression of SAP97 drove GluR1 to synapses, potentiated AMPA receptor (AMPAR) excitatory postsynaptic currents (EPSCs), and occluded LTP. Synaptic potentiation and GluR1 delivery were dissociable by L27 domain mutants that inhibit multimerization of SAP97. Loss of potentiation was correlated with faster turnover of monomeric SAP97 mutants in dendritic spines. We propose that L27-mediated interactions of SAP97 with itself or other proteins regulate the synaptic delivery of AMPARs. RNAi knockdown of endogenous PSD-95 depleted surface GluR1 and impaired AMPA EPSCs. In contrast, RNAi knockdown of endogenous SAP97 reduced surface expression of both GluR1 and GluR2 and inhibited both AMPA and NMDA EPSCs. Thus SAP97 has a broader role than its close relative, PSD-95, in the maintenance of synaptic function.     

Different segmental flexibility of human serum transferrin and lactoferrin

X-ray diffraction studies show that the diferric (holo) forms of human serum transferrin and lactoferrin have almost the same conformation in crystal. In solution, however, the two proteins exhibit different characteristics. The differences are even more pronounced in the apo forms. Small-angle X-ray and neutron scattering data show that lactoferrin is less compact, in apo and holo forms, than the corresponding forms of transferrin in solution. The comparison of primary structures of the two proteins suggests that one of the interdomain hinge regions is significantly longer in lactoferrin than its counterpart in transferrin. The difference in flexibility due to the long hinge region in lactoferrin may be responsible for many of the differences in the physicochemical characteristics of the two proteins.     

Structural characterization of HBXIP: the protein that interacts with the anti-apoptotic protein survivin and the oncogenic viral protein HBx

Hepatitis B X-interacting protein (HBXIP) is a ubiquitous protein that was originally identified as a binding partner of the hepatitis B viral protein HBx. HBXIP is also thought to serve as an anti-apoptotic cofactor of survivin, promoting the suppression of pro-caspase-9 activation. Here we report the crystal structure of the shortest isoform of HBXIP (91 aa long, ∼ 11 kDa) at 1.5 Å resolution. HBXIP crystal shows a monomer per asymmetric unit, with a profilin-like fold which is common to a superfamily of proteins, the Roadblock/LC7 domain family involved in protein-protein interactions. Based on this fold, we propose that HBXIP can form a dimer that can indeed be found in the crystal when symmetric molecules are generated around the asymmetric unit. This dimer shows an extended β-sheet area formed by 10 anti-parallel β-strands from both subunits. Another interesting aspect of the proposed HBXIP dimer interface is the presence of a small leucine zipper between the two α2 helices of each monomer. In solution, the scattering curve obtained by small-angle X-ray scattering for the sample used for crystallization indicates that the protein is predominantly in dimeric form in solution. The fit between the experimental small-angle X-ray scattering curve and the backcalculated curves for two potential crystal dimers shows a significant preference for the Roadblock/LC7 fold dimer model. Moreover, the HBXIP crystal structure represents a step towards understanding the cellular role of HBXIP.