HJURP binds CENP-A via a highly conserved N-terminal domain and mediates its deposition at centromeres

The human histone H3 variant, CENP-A, replaces the conventional histone H3 in centromeric chromatin and, together with centromere-specific DNA-binding factors, directs the assembly of the kinetochore. We purified the prenucelosomal e-CENP-A complex. We found that HJURP, a member of the complex, was required for cell cycle specific targeting of CENP-A to centromeres. HJURP facilitated efficient deposition of CENP-A/H4 tetramers to naked DNA in vitro. Bacterially expressed HJURP binds at a stoichiometric ratio to the CENP-A/H4 tetramer but not to the H3/H4 tetramer. The binding occurred through a conserved HJURP short N-terminal domain, termed CBD. The novel characteristic identified in vertebrates that we named TLTY box of CBD, was essential for formation of the HJURP-CENP-A/H4 complex. Our data identified HJURP as a vertebrate CENP-A chaperone and dissected its mode of interactions with CENP-A.     

FlhA provides the adaptor for coordinated delivery of late flagella building blocks to the type III secretion system

Flagella are the bacterial organelles of motility and can play important roles in pathogenesis. Flagella biosynthesis requires the coordinated export of huge protein amounts from the cytosol to the nascent flagellar structure at the cell surface and employs a type III secretion system (T3SS). Here we show that the integral membrane protein FlhA from the gram-positive bacterium Bacillus subtilis acts as an adaptor for late export substrates at the T3SS. The major filament protein (flagellin) and the filament-cap protein (FliD) bind to the FlhA cytoplasmic domain (FlhA-C) only in complex with their cognate chaperones (FliS and FliT). To understand the molecular details of these interactions we determined the FlhA-C crystal structure at 2.3 Å resolution. FlhA-C consists of an N-terminal linker region, three subdomains with a novel fold, and a disordered region essential for the adaptor function. We show that the export protein FliJ associates with the linker region and modulates the binding properties of FlhA-C. While the interaction of FliD/FliT is enhanced, flagellin/FliS is not affected. FliJ also keeps FliT associated with FlhA-C and excess of FliT inhibits binding of FliD/FliT, suggesting that empty FliT chaperones stay associated with FliJ after export of FliD. Taken together, these results allow to propose a model that explains how the T3SS may switch from the stoichiometric export of FliD to the high-throughput secretion of flagellin.     

Site-directed spin labeling reveals a conformational switch in the phosphorylation domain of smooth muscle myosin

We have used site-directed spin labeling and EPR spectroscopy to detect structural changes within the regulatory light chain (RLC) of smooth muscle myosin upon phosphorylation. Smooth muscle contraction is activated by phosphorylation of S19 on RLC, but the structural basis of this process is unknown. There is no crystal structure containing a phosphorylated RLC, and there is no crystal structure for the N-terminal region of any RLC. Therefore, we have prepared single-Cys mutations throughout RLC, exchanged each mutant onto smooth muscle heavy meromyosin, verified normal regulatory function, and used EPR to determine dynamics and solvent accessibility at each site. A survey of spin-label sites throughout the RLC revealed that only the N-terminal region (first 24 aa) shows a significant change in dynamics upon phosphorylation, with most of the first 17 residues showing an increase in rotational amplitude. Therefore, we focused on this N-terminal region. Additional structural information was obtained from the pattern of oxygen accessibility along the sequence. In the absence of phosphorylation, little or no periodicity was observed, suggesting a lack of secondary structural order in this region. However, phosphorylation induced a strong helical pattern (3.6-residue periodicity) in the first 17 residues, while increasing accessibility throughout the first 24 residues. We have identified a domain within RLC, the N-terminal phosphorylation domain, in which phosphorylation increases helical order, internal dynamics, and accessibility. These results support a model in which this disorder-to-order transition within the phosphorylation domain results in decreased head-head interactions, activating myosin in smooth muscle.     

ClpB N-terminal domain plays a regulatory role in protein disaggregation

ClpB/Hsp100 is an ATP-dependent disaggregase that solubilizes and reactivates protein aggregates in cooperation with the DnaK/Hsp70 chaperone system. The ClpB–substrate interaction is mediated by conserved tyrosine residues located in flexible loops in nucleotide-binding domain-1 that extend into the ClpB central pore. In addition to the tyrosines, the ClpB N-terminal domain (NTD) was suggested to provide a second substrate-binding site; however, the manner in which the NTD recognizes and binds substrate proteins has remained elusive. Herein, we present an NMR spectroscopy study to structurally characterize the NTD–substrate interaction. We show that the NTD includes a substrate-binding groove that specifically recognizes exposed hydrophobic stretches in unfolded or aggregated client proteins. Using an optimized segmental labeling technique in combination with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR, the interaction of client proteins with both the NTD and the pore-loop tyrosines in the 580-kDa ClpB hexamer has been characterized. Unlike contacts with the tyrosines, the NTD–substrate interaction is independent of the ClpB nucleotide state and protein conformational changes that result from ATP hydrolysis. The NTD interaction destabilizes client proteins, priming them for subsequent unfolding and translocation. Mutations in the NTD substrate-binding groove are shown to have a dramatic effect on protein translocation through the ClpB central pore, suggesting that, before their interaction with substrates, the NTDs block the translocation channel. Together, our findings provide both a detailed characterization of the NTD–substrate complex and insight into the functional regulatory role of the ClpB NTD in protein disaggregation.The heat shock protein ClpB (Escherichia coli) or Hsp100 (eukaryotes) is the main protein disaggregase in bacteria, yeast, plants, and mitochondria of all eukaryotic cells, and it is essential for cell survival during severe stress (1–4). Recovery of functional proteins from aggregates by ClpB requires the synergistic interaction with a second molecular chaperone, DnaK (1). Through its cochaperone, DnaJ, DnaK initially binds to the aggregates, leading to the exposure of peptide segments that can be recognized by ClpB (5, 6). DnaK then recruits ClpB to the site of aggregation through direct physical interaction (7, 8), transferring the aggregate to ClpB. Using the energy derived from ATP hydrolysis, ClpB unravels the aggregate by threading single polypeptide chains, one at a time, through the central pore of its hexameric ring (9). Once released from the aggregate, the unfolded polypeptides can either refold spontaneously or fold with the help of additional cellular chaperones.Like other Hsp100 proteins, ClpB forms a hexameric ring, with each protomer comprising an N-terminal domain (NTD) and two nucleotide binding domains (NBD1 and NBD2) separated by a unique regulatory coil–coil domain (10) essential for DnaK binding (7, 11) (Fig. 1 A and B). Both NBDs contain Walker A and Walker B motifs that are required for nucleotide binding and hydrolysis (12, 13), respectively, and a highly conserved tyrosine (Y243 in Thermus thermophilus ClpB) that plays a critical role in disaggregation. Each of the conserved tyrosines from a protomer is located in a so-called pore loop (14) (Fig. 1C) and extends into the axial channel to interact directly with positively charged and aromatic residues from the bound substrate (9, 15). Mutating these NBD-1 pore loop tyrosines leads to a partial reduction of the ClpB protein disaggregation activity (9, 15). When this mutation is combined with the deletion of the NTD, the resulting double mutant is completely inactive in substrate disaggregation (16), although each of these ClpB variants alone can reactivate protein aggregates (9, 15). The complete loss in activity only with the ClpB double mutant was suggested to result from overlapping substrate-binding functions for the NBD1 pore tyrosine residues and the ClpB NTD (16).Open in a separate windowFig. 1.Structure and domain organization of the hexameric ClpB chaperone. Domain organization (A) and protomeric structure (B) of the ClpB chaperone [Protein Data Bank (PDB) ID code 1QVR (10)]. The ClpB protomer consists of an N-terminal domain (NTD; green), two nucleotide binding domains (NBD1, NBD2; dark and light blue, respectively), and a coil–coil domain insertion (CCD; yellow). (C) The monomers assemble into a hexamer consisting of three rings formed by NTDs (top ring; green), NBD1-CCD (blue-yellow), and NBD2 enclosing the central pore. The Inset shows a magnified view of the central pore loops of NBD1 with the conserved tyrosines (Y243; represented as red sticks) extending into the axial channel. This model of ClpB hexamers is based on cryo-electron microscopy structures of E. coli ClpB (EMD-2563) (52).The ClpB NTD is a globular, 150-residue α-helical domain connected by an unstructured 17-residue linker to NBD1 (Fig. 1 A and B) (10). Its precise function remains unclear—it is not required for thermotolerance (17), yet it becomes important in vivo when Hsp70 activity is compromised (18, 19). Although it was reported that the NTD is not required for disaggregation of many small aggregates, it is involved in the reactivation of several strongly aggregated proteins (17, 18, 20). Here, we use NMR to structurally characterize its interaction with substrate proteins and to elucidate its functional role in protein disaggregation. Our results demonstrate that the NTD contains a substrate-binding groove that specifically recognizes hydrophobic residues exposed in unfolded or aggregated client proteins. Unlike the case for substrate binding involving the pore loops, the NTD–substrate interaction is independent both of the nucleotide state and conformational changes to ClpB that ATP hydrolysis promotes. Notably, we show that the NTD interaction destabilizes client proteins, priming them for subsequent unfolding and translocation. Finally, mutations in the NTD substrate-binding groove have a dramatic effect on protein translocation through the ClpB central pore, suggesting that NTDs block the translocation channel before interaction with substrates. Together, our findings provide molecular insight into the NTD–substrate complex as well as into the functional role of the NTD in both protein disaggregation and in regulating ClpB activity.     

Allostery in the ferredoxin protein motif does not involve a conformational switch

Regulation of protein function via cracking, or local unfolding and refolding of substructures, is becoming a widely recognized mechanism of functional control. Oftentimes, cracking events are localized to secondary and tertiary structure interactions between domains that control the optimal position for catalysis and/or the formation of protein complexes. Small changes in free energy associated with ligand binding, phosphorylation, etc., can tip the balance and provide a regulatory functional switch. However, understanding the factors controlling function in single-domain proteins is still a significant challenge to structural biologists. We investigated the functional landscape of a single-domain plant-type ferredoxin protein and the effect of a distal loop on the electron-transfer center. We find the global stability and structure are minimally perturbed with mutation, whereas the functional properties are altered. Specifically, truncating the L1,2 loop does not lead to large-scale changes in the structure, determined via X-ray crystallography. Further, the overall thermal stability of the protein is only marginally perturbed by the mutation. However, even though the mutation is distal to the iron–sulfur cluster (∼20 Å), it leads to a significant change in the redox potential of the iron–sulfur cluster (57 mV). Structure-based all-atom simulations indicate correlated dynamical changes between the surface-exposed loop and the iron–sulfur cluster-binding region. Our results suggest intrinsic communication channels within the ferredoxin fold, composed of many short-range interactions, lead to the propagation of long-range signals. Accordingly, protein interface interactions that involve L1,2 could potentially signal functional changes in distal regions, similar to what is observed in other allosteric systems.     

Identification of a hidden strain switch provides clues to an ancient structural mechanism in protein kinases

The protein kinase catalytic domain contains several conserved residues of unknown functions. Here, using a combination of computational and experimental approaches, we show that the function of some of these residues is to maintain the backbone geometry of the active site in a strained conformation. Specifically, we find that the backbone geometry of the catalytically important HRD motif deviates from ideality in high-resolution structures and the strained geometry results in favorable hydrogen bonds with conserved noncatalytic residues in the active site. In particular, a conserved aspartate in the F-helix hydrogen bonds to the strained HRD backbone in diverse eukaryotic and eukaryotic-like protein kinase crystal structures. Mutations that alter this hydrogen-bonding interaction impair catalytic activity in Aurora kinase. Although the backbone strain is present in most active conformations, several inactive conformations lack the strain because of a peptide flip in the HRD backbone. The peptide flip is correlated with loss of hydrogen bonds with the F-helix aspartate as well as with other interactions associated with kinase regulation. Within protein kinases that are regulated by activation loop phosphorylation, the strained residue is an arginine, which coordinates with the activation loop phosphate. Based on analysis of strain across the protein kinase superfamily, we propose a model in which backbone strain co-evolved with conserved residues for allosteric control of catalytic activity. Our studies provide new clues for the design of allosteric protein kinase inhibitors.     

An engineered lipocalin specific for CTLA-4 reveals a combining site with structural and conformational features similar to antibodies

Biomolecular reagents that enable the specific molecular recognition of proteins play a crucial role in basic research as well as medicine. Up to now, antibodies (immunoglobulins) have been widely used for this purpose. Their predominant feature is the vast repertoire of antigen-binding sites that arise from a set of 6 hypervariable loops. However, antibodies suffer from practical disadvantages because of their complicated architecture, large size, and multiple functions. The lipocalins, on the other hand, have evolved as a protein family that primarily serves for the binding of small molecules. Here, we show that an engineered lipocalin, derived from human Lcn2, can specifically bind the T cell coreceptor CTLA-4 as a prescribed protein target with subnanomolar affinity. Crystallographic analysis reveals that its reshaped cup-like binding site, which is formed by 4 variable loops, provides perfect structural complementarity with this “antigen.” Furthermore, comparison with the crystal structure of the uncomplexed engineered lipocalin indicates a pronounced induced-fit mechanism, a phenomenon so far considered typical for antibodies. By recognizing the same epitope on CTLA-4 that interacts with the counterreceptors B7.1/B7.2 on antigen-presenting cells the engineered Lcn2 exhibits strong, cross-species antagonistic activity, as evidenced by biological effects comparable with a CTLA-4-specific antibody. With its proven stimulatory activity on T cells in vivo, the CTLA-4 blocking lipocalin offers potential for immunotherapy of cancer and infectious disease. Beyond that, lipocalins with engineered antigen-binding sites, so-called Anticalins, provide a class of small (≈180 residues), structurally simple, and robust binding proteins with applications in the life sciences in general.     

The FACT Spt16 "peptidase" domain is a histone H3-H4 binding module

The FACT complex is a conserved cofactor for RNA polymerase II elongation through nucleosomes. FACT bears histone chaperone activity and contributes to chromatin integrity. However, the molecular mechanisms behind FACT function remain elusive. Here we report biochemical, structural, and mutational analyses that identify the peptidase homology domain of the Schizosaccharomyces pombe FACT large subunit Spt16 (Spt16-N) as a binding module for histones H3 and H4. The 2.1-A crystal structure of Spt16-N reveals an aminopeptidase P fold whose enzymatic activity has been lost. Instead, the highly conserved fold directly binds histones H3-H4 through a tight interaction with their globular core domains, as well as with their N-terminal tails. Mutations within a conserved surface pocket in Spt16-N or posttranslational modification of the histone H4 tail reduce interaction in vitro, whereas the globular domains of H3-H4 and the H3 tail bind distinct Spt16-N surfaces. Our analysis suggests that the N-terminal domain of Spt16 may add to the known H2A-H2B chaperone activity of FACT by including a H3-H4 tail and H3-H4 core binding function mediated by the N terminus of Spt16. We suggest that these interactions may aid FACT-mediated nucleosome reorganization events.     

Ribosome recycling depends on a mechanistic link between the FeS cluster domain and a conformational switch of the twin-ATPase ABCE1

Despite some appealing similarities of protein synthesis across all phyla of life, the final phase of mRNA translation has yet to be captured. Here, we reveal the ancestral role and mechanistic principles of the newly identified twin-ATPase ABCE1 in ribosome recycling. We demonstrate that the unique iron-sulfur cluster domain and an ATP-dependent conformational switch of ABCE1 are essential both for ribosome binding and recycling. By direct (11) interaction, the peptide release factor aRF1 is shown to synergistically promote ABCE1 function in posttermination ribosome recycling. Upon ATP binding, ABCE1 undergoes a conformational switch from an open to a closed ATP-occluded state, which drives ribosome dissociation as well as the disengagement of aRF1. ATP hydrolysis is not required for a single round of ribosome splitting but for ABCE1 release from the 30S subunit to reenter a new cycle. These results provide a mechanistic understanding of final phases in mRNA translation.     

Apolipoprotein E4 has extensive conformational heterogeneity in lipid-free and lipid-bound forms

The ε4-allele variant of apolipoprotein E (ApoE4) is the strongest genetic risk factor for Alzheimer’s disease, although it only differs from its neutral counterpart ApoE3 by a single amino acid substitution. While ApoE4 influences the formation of plaques and neurofibrillary tangles, the structural determinants of pathogenicity remain undetermined due to limited structural information. Previous studies have led to conflicting models of the C-terminal region positioning with respect to the N-terminal domain across isoforms largely because the data are potentially confounded by the presence of heterogeneous oligomers. Here, we apply a combination of single-molecule spectroscopy and molecular dynamics simulations to construct an atomically detailed model of monomeric ApoE4 and probe the effect of lipid association. Importantly, our approach overcomes previous limitations by allowing us to work at picomolar concentrations where only the monomer is present. Our data reveal that ApoE4 is far more disordered and extended than previously thought and retains significant conformational heterogeneity after binding lipids. Comparing the proximity of the N- and C-terminal domains across the three major isoforms (ApoE4, ApoE3, and ApoE2) suggests that all maintain heterogeneous conformations in their monomeric form, with ApoE2 adopting a slightly more compact ensemble. Overall, these data provide a foundation for understanding how ApoE4 differs from nonpathogenic and protective variants of the protein.

Apolipoprotein E (ApoE) is a 299-amino acid protein involved in lipid transport and cholesterol homeostasis (1, 2) that plays a key role in Alzheimer’s disease (AD). The polymorphic nature of human APOE allows for encoding three variants (ApoE2, ApoE3, and ApoE4) (3) that have dramatic functional differences, even though it is only a single amino acid change that differentiates ApoE3 from ApoE2 (R158C) and ApoE4 (C112R) (4). The most striking example is ApoE4, which is recognized as the major genetic risk factor for AD (5–9), with individuals who are homozygous for the ε4 allele having up to 15-fold higher probability of developing late-onset AD (10, 11). In contrast, ApoE3 appears to have no impact on the progression of AD, while ApoE2 has been proposed to be protective toward the disease (12). A current hypothesis is that these functional differences stem from structural changes imposed upon ApoE by this single residue substitution and thus having a potential impact on its interaction with AD factors, such as amyloid-beta plaques and neurofibrillary tangles (13, 14). In both the cardiovascular and the central nervous systems, ApoE is prevalently associated noncovalently with lipids as part of lipoproteins, and the single residue substitutions are known to alter its interaction with specific lipoprotein populations (15). From a biochemical point of view, previous work from Garai et al. suggests that only the monomeric form—not the oligomers—is competent for high-affinity lipid binding (16). Therefore, understanding the monomeric structure of ApoE is key to unmasking the mechanisms controlling its interaction with lipids. In addition, recent experiments have found that ApoE expressed by microglia and astrocytes can also occur in poorly lipidated and nonlipidated forms (17). However, a structural characterization of monomeric ApoE in its lipid-free states remains elusive. One major obstacle is posed by the high propensity of ApoE to form oligomers (18), which hampers the investigation of the monomeric form (SI Appendix, Fig. S1). A second challenge is the disordered nature of numerous short segments of the protein, which have been proposed to be flexible and confer structural heterogeneity (19) rendering these regions invisible to conventional structural biology methods.ApoE comprises four different regions: the N-terminal tail (residues 1 to 23), the four-helix bundle (24 to 167) (20–22), the hinge region (168 to 205), and the C-terminal domain (206 to 299) (Fig. 1). Current conformational models (19, 23) of the monomeric lipid-free ApoE agree on the structure of the four-helix bundle (20–22), but they disagree on the configurations of the hinge and C-terminal regions and their orientation with respect to the four-helix bundle. Ensemble Förster resonance energy transfer (FRET) and Electron Paramagnetic Resonance (EPR) studies (24) suggest that ApoE4 forms a close contact between the four-helix bundle and the C-terminal domain, whereas ApoE3 explores more open conformations. This is at odds with the compact set of structures determined by Nuclear Magnetic Resonance (NMR) on a monomeric ApoE3-like variant (22). Recent Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) experiments identified isoform-dependent differences in solvent accessibility of the four-helix bundle, hinting that single amino acid substitutions affect the ability of the C-terminal domain to shield specific regions of the four-helix bundle (19). However, the interpretation of ensemble FRET, EPR (24), and HDX-MS experiments (19) is complicated by the fact that measurements were performed under conditions in which the protein is a stable tetramer (16, 19) and, therefore, are not representative of the conformations of the protein in its monomeric form. The same limitation applies to previous investigations of the folding stability of the protein domains (16, 25–27) and its interaction with lipids (24–29), where ApoE was studied at concentrations that favor either dimer or tetramer conformations (16, 24, 28, 29).Open in a separate windowFig. 1.Protein structural regions and single-molecule constructs of full-length ApoE4. (A) Schematic representation of the secondary structure content in ApoE4 based on the NMR structure (Protein Data Bank (PDB): 2L7B) of the ApoE3-like variant with corresponding designations and identification of the major protein domains: N-terminal tail (gray), four-helix bundle (teal), hinge region (green), and C-terminal domain (light purple). Helical notations are reported for each helix. Labeling positions are identified on the linear sequence by green and red dots (the color scheme is only indicative of FRET labels and not of residue labeling for a specific fluorophore). Yellow dots identify the mutations associated with ApoE3 and ApoE2 variants. Position A86C is located in the random coil between helices H2 and H3 as previously defined (20, 30) and serves as a common reference point to investigate the folded N-terminal domain from two different perspectives. When paired with position A5C (ApoE4_5,86), which is situated upstream of the start of the H1 helix, A86C monitors the conformational properties and folding stability of the N-terminal tail. When paired with position G165C (ApoE4_86,165), which is located at the end of the H4 helix, A86C provides a readout for the folding of the four-helix bundle (22, 30). Positions G182C and A241C (ApoE4_182,241) allow monitoring the behavior of the hinge domain with respect to the C terminus, while positions S223C and A291C (ApoE4_223,291) provide information on the structural properties of the C-terminal domain. Finally, probe positions located at A86C and A241C (ApoE4_86,241) allow us to monitor long-range interactions between the N- and C-terminal domains. (B) One hundred and eighty-degree rotated views of the monomeric ApoE3-like variant NMR structure (PDB: 2L7B) highlighting labeling positions shown in orange. Structure color differentiates the major protein domains described in A.Here, we circumvent these experimental difficulties by harnessing single-molecule fluorescence spectroscopy, an approach that enables working at sufficiently low protein concentrations to avoid oligomerization and directly access the protein in its monomeric form (SI Appendix, Fig. S1). Single-molecule FRET provides a direct readout on the conformations and stability of specific domains within full-length ApoE4 in both the lipid-free and lipid-bound states. We further complement single-molecule observations with molecular dynamics (MD) simulations to obtain an atomically detailed representation of protein conformations that is consistent with our experimental data.     

Directed selection of a conformational antibody domain that prevents mature amyloid fibril formation by stabilizing Abeta protofibrils

The formation of amyloid fibrils is a common biochemical characteristic that occurs in Alzheimer's disease and several other amyloidoses. The unifying structural feature of amyloid fibrils is their specific type of beta-sheet conformation that differentiates these fibrils from the products of normal protein folding reactions. Here we describe the generation of an antibody domain, termed B10, that recognizes an amyloid-specific and conformationally defined epitope. This antibody domain was selected by phage-display from a recombinant library of camelid antibody domains. Surface plasmon resonance, immunoblots, and immunohistochemistry show that this antibody domain distinguishes Abeta amyloid fibrils from disaggregated Abeta peptide as well as from specific Abeta oligomers. The antibody domain possesses functional activity in preventing the formation of mature amyloid fibrils by stabilizing Abeta protofibrils. These data suggest possible applications of B10 in the detection of amyloid fibrils or in the modulation of their formation.     

Variations in the colchicine-binding domain provide insight into the structural switch of tubulin

Structural changes occur in the αβ-tubulin heterodimer during the microtubule assembly/disassembly cycle. Their most prominent feature is a transition from a straight, microtubular structure to a curved structure. There is a broad range of small molecule compounds that disturbs the microtubule cycle, a class of which targets the colchicine-binding site and prevents microtubule assembly. This class includes compounds with very different chemical structures, and it is presently unknown whether they prevent tubulin polymerization by the same mechanism. To address this issue, we have determined the structures of tubulin complexed with a set of such ligands and show that they interfere with several of the movements of tubulin subunits structural elements upon its transition from curved to straight. We also determined the structure of tubulin unliganded at the colchicine site; this reveals that a β-tubulin loop (termed T7) flips into this site. As with colchicine site ligands, this prevents a helix which is at the interface with α-tubulin from stacking onto a β-tubulin β sheet as in straight protofilaments. Whereas in the presence of these ligands the interference with microtubule assembly gets frozen, by flipping in and out the β-subunit T7 loop participates in a reversible way in the resistance to straightening that opposes microtubule assembly. Our results suggest that it thereby contributes to microtubule dynamic instability.     

Probing large conformational rearrangements in wild-type and mutant spectrin using structural mass spectrometry

Conformational changes of macromolecular complexes play key mechanistic roles in many biological processes, but large, highly flexible proteins and protein complexes usually cannot be analyzed by crystallography or NMR. Here, structures and conformational changes of the highly flexible, dynamic red cell spectrin and effects of a common mutation that disrupts red cell membranes were elucidated using chemical cross-linking coupled with mass spectrometry. Interconversion of spectrin between closed dimers, open dimers, and tetramers plays a key role in maintaining red cell shape and membrane integrity, and spectrins in other cell types serve these as well as more diverse functions. Using a minispectrin construct, experimentally verified structures of closed dimers and tetramers were determined by combining distance constraints from zero-length cross-links with molecular models and biophysical data. Subsequent biophysical and structural mass spectrometry characterization of a common hereditary elliptocytosis-related mutation of α-spectrin, L207P, showed that cell membranes were destabilized by a shift of the dimer–tetramer equilibrium toward closed dimers. The structure of αL207P mutant closed dimers provided previously unidentified mechanistic insight into how this mutation, which is located a large distance from the tetramerization site, destabilizes spectrin tetramers and cell membrane integrity.Solving static structures of protein complexes and probing dynamic conformational rearrangements have frequently provided mechanistic insights into macromolecular functions as well as effects of disease-related mutations. However, high-resolution structural techniques such as X-ray crystallography and NMR usually cannot be applied to large proteins that are highly flexible, intrinsically disordered, or undergo large conformational changes. Chemical cross-linking coupled with mass spectrometry (CX-MS) is a powerful tool that identifies proximal amino acid residues of proteins in solution. These spatial constraints can greatly enhance and experimentally validate molecular modeling to result in reliable medium-resolution structures. This approach has been effectively used in numerous studies (1–5), although homobifunctional lysine-specific cross-linkers with relatively long spacer arms were mostly used. Such reagents have been preferred because they enable introduction of isotope labels and other functional sites that facilitate cross-linked peptide identifications (6–9). In contrast, zero-length cross-linkers such as 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide form a covalent bond between reactive amines (N-terminal amine or lysine side chain) and carboxyls (C-terminal carboxyl or aspartic or glutamic acid side chains) without inserting extra atoms (Fig. 1). Hence, reactive groups have to be within salt bridge distances to react. This results in tighter distance constraints that outperform those from longer cross-links when these data are used to refine structural models, and a much lower density of cross-links is needed to achieve high-quality structures (7). In this study we used a recently developed strategy for in-depth identification of zero-length cross-links, as summarized in Fig. 1, to probe structures and conformational changes in spectrin, a highly flexible, dynamic protein with multiple functions that is typically associated with cell membranes. In red cells, spectrin is the central component of a highly specialized, 2D, net-like submembraneous complex that confers both structural integrity and elasticity to the cell membrane. The 1,052-kDa spectrin tetramer is a long, highly flexible, worm-like protein composed primarily of many tandem, homologous, “spectrin-type” domains (Fig. 2A). In all reported crystal structures, these domains are approximately 50-Å-long three-helix bundles with helical connectors (Fig. 2 B and C). To date, crystallization of more than four spectrin-type domains has not been feasible, presumably owing to spectrin’s highly flexible nature. In the membrane skeleton, spectrin tetramers bridge short actin oligomers, and membrane stability is highly dependent upon the dimer–tetramer equilibrium because conversion to dimers breaks the spectrin bridges between actin oligomers (10, 11).Open in a separate windowFig. 1.Schematic for the zero-length CX-MS data acquisition and data analysis pipeline.Open in a separate windowFig. 2.Spectrin topography and minispectrin tetramer structure. (A) Schematic showing spectrin domains, the dimer–tetramer equilibria, and minispectrin. The spectrin-type domains that constitute most of the molecule are represented as rounded rectangles. Red asterisks in the minispectrin cartoon indicate the approximate location of the αL207P mutation. (B) Superimposition of the four crystal structures of spectrin-type domains [Protein Data Bank (PBD) ID: 1CUN, 1U5P, 3FB2, and 1S35] used as template building blocks for homology modeling. (C) Crystal structure for the spectrin tetramerization interface (PDB ID: 3LBX). (D) Locations of interdomain cross-links used to model minispectrin tetramer; blue lines, cross-links identified previously (21); red lines, previously unidentified cross-links; dashed lines, the same cross-links repeated in the second half of the tetramer. (E) Superimposition of present and previous tetramer structures. (F) Space-filling representations of tetramer models. β-Spectrin domains are colored in bright or pale cyan, and α-spectrin domains are colored in bright or pale orange to distinguish the two strands.The spectrin dimer–tetramer equilibrium actually involves three states, including closed dimers, open dimers, and bivalent head-to-head tetramers (Fig. 2A). Conversion from open to closed dimers involves a large conformational rearrangement of the longer α-subunit, where it folds back upon itself and forms a head-to-head association analogous to the head-to-head associations in tetramers (Fig. 2A). Closed dimers play a critical but poorly understood role in this equilibrium and are responsible for a high-energy threshold that regulates kinetics of the dimer–tetramer equilibrium (12). Furthermore, hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP) are common human clinical disorders characterized by reduced spectrin tetramerization resulting in abnormal red cell shape, increased membrane fragility, and in some cases, severe anemia that is transfusion dependent (13, 14). Many HE and HPP mutations are located within the tetramerization site (13), although a number of interesting mutations are located large distances from this binding domain and destabilize tetramer formation through unknown mechanisms (15–19). For example, the very common αL207P mutation is located in the middle of the α2 domain, which is ∼75 Å from the tetramerization site. However, it seemed likely that this region could undergo important conformational changes during the closed–open dimer transition (Fig. 2A). We previously developed a 90 kDa fused minispectrin dimer (Fig. 2A) to further study the dimer–tetramer equilibrium (20) and used it here for the CX-MS experiments because it is substantially simpler than the 526-kDa full-length spectrin dimer and retains physiological tetramer binding properties.     

Structures of P-glycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain

P-glycoprotein (P-gp) is one of the best-known mediators of drug efflux-based multidrug resistance in many cancers. This validated therapeutic target is a prototypic, plasma membrane resident ATP-Binding Cassette transporter that pumps xenobiotic compounds out of cells. The large, polyspecific drug-binding pocket of P-gp recognizes a variety of structurally unrelated compounds. The transport of these drugs across the membrane is coincident with changes in the size and shape of this pocket during the course of the transport cycle. Here, we present the crystal structures of three inward-facing conformations of mouse P-gp derived from two different crystal forms. One structure has a nanobody bound to the C-terminal side of the first nucleotide-binding domain. This nanobody strongly inhibits the ATP hydrolysis activity of mouse P-gp by hindering the formation of a dimeric complex between the ATP-binding domains, which is essential for nucleotide hydrolysis. Together, these inward-facing conformational snapshots of P-gp demonstrate a range of flexibility exhibited by this transporter, which is likely an essential feature for the binding and transport of large, diverse substrates. The nanobody-bound structure also reveals a unique epitope on P-gp.     

Structural basis for intersubunit signaling in a protein disaggregating machine

ClpB is a ring-forming, ATP-dependent protein disaggregase that cooperates with the cognate Hsp70 system to recover functional protein from aggregates. How ClpB harnesses the energy of ATP binding and hydrolysis to facilitate the mechanical unfolding of previously aggregated, stress-damaged proteins remains unclear. Here, we present crystal structures of the ClpB D2 domain in the nucleotide-bound and -free states, and the fitted cryoEM structure of the D2 hexamer ring, which provide a structural understanding of the ATP power stroke that drives protein translocation through the ClpB hexamer. We demonstrate that the conformation of the substrate-translocating pore loop is coupled to the nucleotide state of the cis subunit, which is transmitted to the neighboring subunit via a conserved but structurally distinct intersubunit-signaling pathway common to diverse AAA+ machines. Furthermore, we found that an engineered, disulfide cross-linked ClpB hexamer is fully functional biochemically, suggesting that ClpB deoligomerization is not required for protein disaggregation.     

Defining a length scale for millisecond-timescale protein conformational exchange

Although atomic resolution 3D structures of protein native states and some folding intermediates are available, the mechanism of interconversion between such states remains poorly understood. Here we study the four-helix bundle FF module, which folds via a transiently formed and sparsely populated compact on-pathway intermediate, I. Relaxation dispersion NMR spectroscopy has previously been used to elucidate the 3D structure of this intermediate and to establish that the conformational exchange between the I and the native, N, states of the FF domain is driven predominantly by water dynamics. In the present study we use NMR methods to define a length scale for the FF I–N transition, namely the effective hydrodynamic radius (EHR) that provides an average measure of the size of the structural units participating in the transition at any given time. Our experiments establish that the EHR is less than 4 Å, on the order of the size of one to two amino acid side chains, much smaller than the FF domain hydrodynamic radius (13 Å). The small magnitude of the EHR provides strong evidence that the I–N interconversion does not proceed via the synchronous motion of large clusters of amino acid residues, but rather by the exposure/burial of one or two side chains from solvent at any given time. Because the hydration of small hydrophobic solutes (< 4 Å) does not involve considerable dewetting or disruption of the water–hydrogen bonding network, the FF domain I–N transition does not require appreciable changes to the structure of the surrounding water.