Acetylation of L12 increases interactions in the Escherichia coli ribosomal stalk complex

The ribosomal stalk complex in Escherichia coli consists of L10 and four copies of L7/L12, and is largely responsible for binding and recruiting translation factors. Structural characterisation of this stalk complex is difficult, primarily due to its dynamics. Here, we apply mass spectrometry to follow post-translational modifications and their effect on structural changes of the stalk proteins on intact ribosomes. Our results show that increased acetylation of L12 occurs during the stationary phase on ribosomes harvested from cells grown under optimal conditions. For cells grown in minimal medium, L12 acetylation and processing is altered, resulting in deficient removal of N-terminal methionine in ∼ 50% of the L12 population, while processed L12 is almost 100% acetylated. Our results show also that N-acetylation of L12 correlates with an increased stability of the stalk complex in the gas phase. To investigate further the basis of this increased stability, we applied a solution phase hydrogen deuterium exchange protocol to compare the rate of deuterium incorporation in the proteins L9, L10, L11 and L12 as well as the acetylated form of L12 (L7), in situ on the ribosome. Results show that deuterium incorporation is consistently slower for L7 relative to L12 and for L10 when L7 is predominant. Our results imply a tightening of the interaction between L7 and L10 relative to that between L12 and L10. Since acetylation is predominant when cells are grown in minimal medium, we propose that these modifications form part of the cell's strategy to increase stability of the stalk complex under conditions of stress. More generally, our results demonstrate that it is possible to discern the influence of a 42 Da post-translational modification by mass spectrometry and to record subtle changes in hydrogen/deuterium exchange within the context of an intact 2.5 MDa particle.     

Dissection of Conformational Conversion Events during Prion Amyloid Fibril Formation Using Hydrogen Exchange and Mass Spectrometry

A molecular understanding of prion diseases requires an understanding of the mechanism of amyloid fibril formation by the prion protein. In particular, it is necessary to define the sequence of the structural events describing the conformational conversion of monomeric PrP to aggregated PrP. In this study, the sequence of the structural events in the case of amyloid fibril formation by recombinant mouse prion protein at pH 7 has been characterized by hydrogen–deuterium exchange and mass spectrometry. The observation that fibrils are substantially more stable to hydrogen–deuterium exchange than is native monomer allows both forms to be quantified during the course of the aggregation reaction. Under the aggregation conditions utilized, native monomeric protein and amyloid fibrils are the only forms of the protein detectable during the course of the fibril formation reaction, suggesting that monomer directly adds on to the fibril template. Conformational conversion is shown to occur in two steps after the binding of monomer to fibril, with helix 1 unfolding only after helices 2 and 3 transform into β-sheet. Local stability in the β-sheet core region (residues ~ 159–225) of the fibrils is shown to be sequence dependent in that it varies along the length of the core, and local stability in protein molecules that are ordered in the structurally heterogeneous sequence segment 109–132 is shown to be similar to that in the core. This new understanding of the structural events during prion protein aggregation has important bearing on our comprehension of the molecular basis of prion pathogenesis.     

Nucleotide- and Activator-Dependent Structural and Dynamic Changes of Arp2/3 Complex Monitored by Hydrogen/Deuterium Exchange and Mass Spectrometry

Arp2/3 complex plays a central role in the de novo nucleation of filamentous actin as branches on existing filaments. The complex must bind ATP, protein activators [e.g., Wiskott-Aldrich syndrome protein (WASp)], and the side of an actin filament to form a new actin filament. Amide hydrogen/deuterium exchange coupled with mass spectrometry was used to examine the structural and dynamic properties of the mammalian Arp2/3 complex in the presence of both ATP and the activating peptide segment from WASp. Changes in the rate of hydrogen exchange indicate that ATP binding causes conformational rearrangements of Arp2 and Arp3 that are transmitted allosterically to the Arp complex (ARPC)1, ARPC2, ARPC4, and ARPC5 subunits. These data are consistent with the closure of nucleotide-binding cleft of Arp3 upon ATP binding, resulting in structural rearrangements that propagate throughout the complex. Binding of the VCA domain of WASp to ATP-Arp2/3 further modulates the rates of hydrogen exchange in these subunits, indicating that a global conformational reorganization is occurring. These effects may include the direct binding of activators to Arp3, Arp2, and ARPC1; alterations in the relative orientations of Arp2 and Arp3; and the long-range transmission of activator-dependent signals to segments proposed to be involved in binding the F-actin mother filament.     

Rapid Analysis of Protein Structure and Dynamics by Hydrogen/Deuterium Exchange Mass Spectrometry

An automated approach for the rapid analysis of protein structure has been developed and used to study acid-induced conformational changes in human growth hormone. The labeling approach involves hydrogen/deuterium exchange (H/D-Ex) of protein backbone amide hydrogens with rapid and sensitive detection by mass spectrometry (MS). Briefly, the protein is incubated for defined intervals in a deuterated environment. After rapid quenching of the exchange reaction, the partially deuterated protein is enzymatically digested and the resulting peptide fragments are analyzed by liquid chromatography mass spectrometry (LC-MS). The deuterium buildup curve measured for each fragment yields an average amide exchange rate that reflects the environment of the peptide in the intact protein. Additional analyses allow mapping of the free energy of folding on localized segments along the protein sequence affording unique dynamic and structural information. While amide H/D-Ex coupled with MS is recognized as a powerful technique for studying protein structure and protein–ligand interactions, it has remained a labor-intensive task. The improvements in the amide H/D-Ex methodology described here include solid phase proteolysis, automated liquid handling and sample preparation, and integrated data reduction software that together improve sequence coverage and resolution, while achieving a sample throughput nearly 10-fold higher than the commonly used manual methods.     

Time-Dependent Changes in Side-Chain Solvent Accessibility during Cytochrome c Folding Probed by Pulsed Oxidative Labeling and Mass Spectrometry

The current work employs a novel approach for characterizing structural changes during the refolding of acid-denatured cytochrome c (cyt c). At various time points (ranging from 10 ms to 5 min) after a pH jump from 2 to 7, the protein is exposed to a microsecond hydroxyl radical (·OH) pulse that induces oxidative labeling of solvent-exposed side chains. Most of the covalent modifications appear as + 16-Da adducts that are readily detectable by mass spectrometry. The overall extent of labeling decreases as folding proceeds, reflecting dramatic changes in the accessibility of numerous residues. Peptide mapping and tandem mass spectrometry reveal that the side chains of C14, C17, H33, F46, Y48, W59, M65, Y67, Y74, M80, I81, and Y97 are among the dominant sites of oxidation. Temporal changes in the accessibility of these residues are consistent with docking of the N- and C-terminal helices as early as 10 ms. However, structural reorganization at the helix interface takes place up to at least 1 s. Initial misligation of the heme iron by H33 leads to distal crowding, giving rise to low solvent accessibility of the displaced (native) M80 ligand and the adjacent I81. W59 retains a surprisingly high level of accessibility long into the folding process, indicating the presence of packing defects in the hydrophobically collapsed core. Overall, the results of this work are consistent with previous hydrogen/deuterium exchange studies that proposed a foldon-mediated mechanism. The structural data obtained by ·OH labeling monitor the packing and burial of side chains, whereas hydrogen/deuterium exchange primarily monitors the formation of secondary structure elements. Hence, the two approaches yield complementary information. Considering the very short time scale of pulsed oxidative labeling, an extension of the approach used here to sub-millisecond folding studies should be feasible.     

Activation of ClpP Protease by ADEP Antibiotics: Insights from Hydrogen Exchange Mass Spectrometry

The bacterial protease ClpP consists of 14 subunits that assemble into two stacked heptameric rings. The central degradation chamber can be accessed via axial pores. In free ClpP, these pores are obstructed by the N-terminal regions of the seven subunits at either end of the barrel. Acyldepsipeptides (ADEPs) are antibacterial compounds that bind in hydrophobic clefts surrounding the pore region, causing the pores to open up. The ensuing uncontrolled degradation of intracellular proteins is responsible for the antibiotic activity of ADEPs. Recently published X-ray structures yielded conflicting models regarding the conformation adopted by the N-terminal regions in the open state. Here, we use hydrogen/deuterium exchange (HDX) mass spectrometry to obtain complementary insights into the ClpP behavior with and without ADEP1. Ligand binding causes rigidification of the equatorial belt, accompanied by destabilization in the vicinity of the binding clefts. The N-terminal regions undergo rapid deuteration with only minor changes after ADEP1 binding, revealing a lack of stable H-bonding. Our data point to a mechanism where the pore opening mechanism is mediated primarily by changes in the packing of N-terminal nonpolar side chains. We propose that a “hydrophobic plug” causes pore blockage in ligand-free ClpP. ADEP1 binding provides new hydrophobic anchor points that nonpolar N-terminal residues can interact with. In this way, ADEP1 triggers the transition to an open conformation, where nonpolar moieties are clustered around the rim of the pore. This proposed mechanism helps reconcile the conflicting models that had been put forward earlier.     

Conformational dynamics of the molecular chaperone Hsp90 in complexes with a co-chaperone and anticancer drugs

The molecular chaperone Hsp90 is essential for the correct folding, maturation and activation of a diverse array of client proteins, including several key constituents of oncogenic processes. Hsp90 has become a focus of cancer research, since it represents a target for direct prophylaxis against multistep malignancy. Hydrogen-exchange mass spectrometry was used to study the structural and conformational changes undergone by full-length human Hsp90beta in solution upon binding of the kinase-specific co-chaperone Cdc37 and two Hsp90 ATPase inhibitors: Radicicol and the first-generation anticancer drug DMAG. Changes in hydrogen exchange pattern in the complexes in regions of Hsp90 remote to the ligand-binding site were observed indicating long-range effects. In particular, the interface between the N-terminal domain and middle domains exhibited significant differences between the apo and complexed forms. For the inhibitors, differences in the interface between the middle domain and the C-terminal domain were also observed. These data provide important insight into the structure of the biologically active form of the protein.     

Phosphorylation-Induced Activation of the Response Regulator VraR from Staphylococcus aureus: Insights from Hydrogen Exchange Mass Spectrometry

A two-component system consisting of the histidine kinase vancomycin-resistance-associated sensor and the response regulator vancomycin-resistance-associated regulator (VraR) allows Staphylococcus aureus to sense antibiotic-related cell wall stress and to mount a suitable response. An experimental structure of full-length VraR is not available yet, but previous work points to similarities between VraR and the well-characterized NarL. This work employs hydrogen exchange mass spectrometry to gain insights into the phosphorylation-induced activation of VraR, a process that primes the protein for dimerization and DNA binding. Whereas VraR is highly dynamic, phosphorylated VraR shows less extensive deuteration. This rigidification is most dramatic within the receiver domain, which carries the phosphorylation site D55. Alterations in the DNA-binding domain are much less pronounced. Changes in deuteration within the receiver domain are consistent with a Y-T coupling mechanism. In analogy to NarL, the activation of VraR is thought to involve separation and subsequent reorientation of the two domains, thereby allowing the α8-turn-α9 element to engage in DNA binding. The current work suggests that this structural transition is triggered by a reduction in the effective length of the linker through enhanced hydrogen bonding. In addition, separation of the two domains may be favored by the establishment of noncovalent protein-protein interactions and intradomain contacts at the expense of previously existing interdomain bonds. α9 appears to be packed against the receiver domain in nonactivated VraR. Support is presented for α1 as a dimerization interface in phosphorylated VraR, whereas protein-protein interactions for nonphosphorylated VraR are impeded by extensive disorder in this region.     

Mass spectrometry of hydrogen/deuterium exchange in 70S ribosomal proteins from E. coli

The 70S ribosome from Escherichia coli is a supermacro complex (MW: 2.7MDa) comprising three RNA molecules and more than 50 proteins. We have for the first time successfully analyzed the flexibility of 70S ribosomal proteins in solution by detecting the hydrogen/deuterium exchange with mass spectrometry. Based on the deuterium incorporation map of the X-ray structure obtained at the time of each exchange, we demonstrate the structure-flexibility-function relationship of ribosome focusing on the deuterium incorporation of the proteins binding ligands (tRNA, mRNA, and elongation factor) and the relation with structural assembly processes.     

Structure and Dynamics of NBD1 from CFTR Characterized Using Crystallography and Hydrogen/Deuterium Exchange Mass Spectrometry

The ΔF508 mutation in nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the predominant cause of cystic fibrosis. Previous biophysical studies on human F508 and ΔF508 domains showed only local structural changes restricted to residues 509-511 and only minor differences in folding rate and stability. These results were remarkable because ΔF508 was widely assumed to perturb domain folding based on the fact that it prevents trafficking of CFTR out of the endoplasmic reticulum. However, the previously reported crystal structures did not come from matched F508 and ΔF508 constructs, and the ΔF508 structure contained additional mutations that were required to obtain sufficient protein solubility. In this article, we present additional biophysical studies of NBD1 designed to address these ambiguities. Mass spectral measurements of backbone amide ¹H/²H exchange rates in matched F508 and ΔF508 constructs reveal that ΔF508 increases backbone dynamics at residues 509-511 and the adjacent protein segments but not elsewhere in NBD1. These measurements also confirm a high level of flexibility in the protein segments exhibiting variable conformations in the crystal structures. We additionally present crystal structures of a broader set of human NBD1 constructs, including one harboring the native F508 residue and others harboring the ΔF508 mutation in the presence of fewer and different solubilizing mutations. The only consistent conformational difference is observed at residues 509-511. The side chain of residue V510 in this loop is mostly buried in all non-ΔF508 structures but completely solvent exposed in all ΔF508 structures. These results reinforce the importance of the perturbation ΔF508 causes in the surface topography of NBD1 in a region likely to mediate contact with the transmembrane domains of CFTR. However, they also suggest that increased exposure of the 509-511 loop and increased dynamics in its vicinity could promote aggregation in vitro and aberrant intermolecular interactions that impede trafficking in vivo.     

Allosteric signaling in the biotin repressor occurs via local folding coupled to global dampening of protein dynamics

The biotin repressor is an allosterically regulated, site-specific DNA-binding protein. Binding of the small ligand bio-5′-AMP activates repressor dimerization, which is a prerequisite to DNA binding. Multiple disorder-to-order transitions, some of which are known to be important for the functional allosteric response, occur in the vicinity of the ligand-binding site concomitant with effector binding to the repressor monomer. In this work, the extent to which these local changes are coupled to additional changes in the structure/dynamics of the repressor was investigated using hydrogen/deuterium exchange coupled to mass spectrometry. Measurements were performed on the apo-protein and on complexes of the protein bound to four different effectors that elicit a range of thermodynamic responses in the repressor. Global exchange measurements indicate that binding of any effector to the intact protein is accompanied by protection from exchange. Mass spectrometric analysis of pepsin-cleavage products generated from the exchanged complexes reveals that the protection is distributed throughout the protein. Furthermore, the magnitude of the level of protection in each peptide from hydrogen/deuterium exchange correlates with the magnitude of the functional allosteric response elicited by a ligand. These results indicate that local structural changes in the binding site that occur concomitant with effector binding nucleate global dampening of dynamics. Moreover, the magnitude of dampening of repressor dynamics tracks with the magnitude of the functional response to effector binding.     

Conformational Dynamics of the Bovine Mitochondrial ADP/ATP Carrier Isoform 1 Revealed by Hydrogen/Deuterium Exchange Coupled to Mass Spectrometry

The mitochondrial adenine nucleotide carrier (Ancp) catalyzes the transport of ADP and ATP across the mitochondrial inner membrane, thus playing an essential role in cellular energy metabolism. During the transport mechanism the carrier switches between two different conformations that can be blocked by two toxins: carboxyatractyloside (CATR) and bongkrekic acid. Therefore, our understanding of the nucleotide transport mechanism can be improved by analyzing structural differences of the individual inhibited states. We have solved the three-dimensional structure of bovine carrier isoform 1 (bAnc1p) in a complex with CATR, but the structure of the carrier-bongkrekic acid complex, and thus, the detailed mechanism of transport remains unknown. Improvements in sample processing in the hydrogen/deuterium exchange technique coupled to mass spectrometry (HDX-MS) have allowed us to gain novel insights into the conformational changes undergone by bAnc1p. This paper describes the first study of bAnc1p using HDX-MS. Results obtained with the CATR-bAnc1p complex were fully in agreement with published results, thus, validating our approach. On the other hand, the HDX kinetics of the two complexes displays marked differences. The bongkrekic acid-bAnc1p complex exhibits greater accessibility to the solvent on the matrix side, whereas the CATR-bAnc1p complex is more accessible on the intermembrane side. These results are discussed with respect to the structural and biochemical data available on Ancp.     

Dynamics and Conformational Changes in Human NEIL2 DNA Glycosylase Analyzed by Hydrogen/Deuterium Exchange Mass Spectrometry

Base excision DNA repair (BER) is necessary for removal of damaged nucleobases from the genome and their replacement with normal nucleobases. BER is initiated by DNA glycosylases, the enzymes that cleave the N-glycosidic bonds of damaged deoxynucleotides. Human endonuclease VIII-like protein 2 (hNEIL2), belonging to the helix–two-turn–helix structural superfamily of DNA glycosylases, is an enzyme uniquely specific for oxidized pyrimidines in non-canonical DNA substrates such as bubbles and loops. The structure of hNEIL2 has not been solved; its closest homologs with known structures are NEIL2 from opossum and from giant mimivirus. Here we analyze the conformational dynamics of free hNEIL2 using a combination of hydrogen/deuterium exchange mass spectrometry, homology modeling and molecular dynamics simulations. We show that a prominent feature of vertebrate NEIL2 – a large insert in its N-terminal domain absent from other DNA glycosylases – is unstructured in solution. It was suggested that helix–two-turn–helix DNA glycosylases undergo open–close transition upon DNA binding, with the large movement of their N- and C-terminal domains, but the open conformation has been elusive to capture. Our data point to the open conformation as favorable for free hNEIL2 in solution. Overall, our results are consistent with the view of hNEIL2 as a conformationally flexible protein, which may be due to its participation in the repair of non-canonical DNA structures and/or to the involvement in functional and regulatory protein–protein interactions.     

Elucidating the Mechanism of Substrate Recognition by the Bacterial Hsp90 Molecular Chaperone

Hsp90 is a conformationally dynamic molecular chaperone known to promote the folding and activation of a broad array of protein substrates (“clients”). Hsp90 is believed to preferentially interact with partially folded substrates, and it has been hypothesized that the chaperone can significantly alter substrate structure as a mechanism to alter the substrate functional state. However, critically testing the mechanism of substrate recognition and remodeling by Hsp90 has been challenging. Using a partially folded protein as a model system, we find that the bacterial Hsp90 adapts its conformation to the substrate, forming a binding site that spans the middle and C-terminal domains of the chaperone. Cross-linking and NMR measurements indicate that Hsp90 binds to a large partially folded region of the substrate and significantly alters both its local and long-range structure. These findings implicate Hsp90's conformational dynamics in its ability to bind and remodel partially folded proteins. Moreover, native-state hydrogen exchange indicates that Hsp90 can also interact with partially folded states only transiently populated from within a thermodynamically stable, native-state ensemble. These results suggest a general mechanism by which Hsp90 can recognize and remodel native proteins by binding and remodeling partially folded states that are transiently sampled from within the native ensemble.     

Functional characterization of the CDF transporter SMc02724 (SmYiiP) in Sinorhizobium meliloti: Roles in manganese homeostasis and nodulation

In bacteria, membrane transporters of the cation diffusion facilitator (CDF) family participate in Zn^2 +, Fe^2 +, Mn^2 +, Co^2 + and Ni^2 + homeostasis. The functional role during infection processes for several members has been shown to be linked to the specificity of transport. Sinorhizobium meliloti has two homologous CDF genes with unknown transport specificity. Here we evaluate the role played by the CDF SMc02724 (SmYiiP). The deletion mutant strain of SmYiiP (ΔsmyiiP) showed reduced in vitro growth fitness only in the presence of Mn^2 +. Incubation of ΔsmyiiP and WT cells with sub-lethal Mn^2 + concentrations resulted in a 2-fold increase of the metal only in the mutant strain. Normal levels of resistance to Mn^2 + were attained by complementation with the gene SMc02724 under regulation of its endogenous promoter. In vitro, liposomes with incorporated heterologously expressed pure protein accumulated several transition metals. However, only the transport rate of Mn^2 + was increased by imposing a transmembrane H⁺ gradient. Nodulation assays in alfalfa plants showed that the strain ΔsmyiiP induced a lower number of nodules than in plants infected with the WT strain. Our results indicate that Mn^2 + homeostasis in S. meliloti is required for full infection capacity, or nodule function, and that the specificity of transport in vivo of SmYiiP is narrowed down to Mn^2 + by a mechanism involving the proton motive force.     

Metal ion and inter-domain interactions as functional networks in E. coli topoisomerase I

Escherichia coli topoisomerase I (EcTopoI) is a type IA bacterial topoisomerase which is receiving large attention due to its potential application as novel target for antibacterial therapeutics. Nevertheless, a detailed knowledge of its mechanism of action at molecular level is to some extent lacking. This is partly due to the requirement of several factors (metal ions, nucleic acid) to the proper progress of the enzyme catalytic cycle. Additionally, each of them can differently affect the protein structure.     

PAS domain allostery and light-induced conformational changes in photoactive yellow protein upon I2 intermediate formation, probed with enhanced hydrogen/deuterium exchange mass spectrometry

Photoactive yellow protein (PYP) is a small bacterial photoreceptor that undergoes a light-activated reaction cycle. PYP is also the prototypical Per-Arnt-Sim (PAS) domain. PAS domains, found in diverse multi-domain proteins from bacteria to humans, mediate protein-protein interactions and function as sensors and signal transducers. Here, we investigate conformational and dynamic changes in solution in wild-type PYP upon formation of the long-lived putative signaling intermediate I2 with enhanced hydrogen/deuterium exchange mass spectrometry (DXMS). The DXMS results showed that the central beta-sheet remains stable but specific external protein segments become strongly deprotected. Light-induced disruption of the dark-state hydrogen bonding network in I2 produces increased flexibility and opening of PAS core helices alpha3 and alpha4, releases the beta4-beta5 hairpin, and propagates conformational changes to the central beta-sheet. Surprisingly, the first approximately 10 N-terminal residues, which are essential for fast dark-state recovery from I2, become more protected. By combining the DXMS results with our crystallographic structures, which reveal detailed changes near the chromophore but limited protein conformational change, we propose a mechanism for I2 state formation. This mechanism integrates the results from diverse biophysical studies of PYP, and links an allosteric T to R-state conformational transition to three pathways for signal propagation within the PYP fold. On the basis of the observed changes in PYP plus commonalities shared among PAS domain proteins, we further propose that PAS domains share this conformational mechanism, which explains the versatile signal transduction properties of the structurally conserved PYP/PAS module by framework-encoded allostery.     

Cloning and functional expression in E. coli of a polyphenol oxidase transcript from Coreopsis grandiflora involved in aurone formation

Polyphenol oxidases are involved in aurone biosynthesis but the gene responsible for 4-deoxyaurone formation in Asteraceae was so far unknown. Three novel full-length cDNA sequences were isolated from Coreopsis grandiflora with sizes of 1.80 kb (cgAUS1) and 1.85 kb (cgAUS2a, 2b), encoding for proteins of 68–69 kDa, respectively. cgAUS1 is preferably expressed in young petals indicating a specific role in pigment formation. The 58.9 kDa AUS1 holoproenzyme, was recombinantly expressed in E. coli and purified to homogeneity. The enzyme shows only diphenolase activity, catalyzing the conversion of chalcones to aurones and was characterized by SDS–PAGE and shot-gun type nanoUHPLC–ESI-MS/MS.