Unexpected Gating Behaviour of an Engineered Potassium Channel Kir.

In this study, we investigated the dynamics and functional characteristics of the KirBac3.1 S129R, a mutated bacterial potassium channel for which the inner pore-lining helix (TM2) was engineered so that the bundle crossing is trapped in an open conformation. The structure of this channel has been previously determined at high atomic resolution. We explored the dynamical characteristics of this open state channel using an in silico method MDeNM that combines molecular dynamics simulations and normal modes. We captured the global and local motions at the mutation level and compared these data with HDX-MS experiments. MDeNM provided also an estimation of the probability of the different opening states that are in agreement with our electrophysiological experiments. In the S129R mutant, the Arg129 mutation releases the two constriction points in the channel that existed in the wild type but interestingly creates another restriction point.

Integrative Study of the Structural and Dynamical Properties of a KirBac3.1 Mutant: Functional Implication of a Highly Conserved Tryptophan in the Transmembrane Domain.

ATP-sensitive potassium (K-ATP) channels are ubiquitously expressed on the plasma membrane of cells in several organs, including the heart, pancreas, and brain, and they govern a wide range of physiological processes. In pancreatic ß-cells, K-ATP channels composed of Kir6.2 and SUR1 play a key role in coupling blood glucose and insulin secretion. A tryptophan residue located at the cytosolic end of the transmembrane helix is highly conserved in eukaryote and prokaryote Kir channels. Any mutation on this amino acid causes a gain of function and neonatal diabetes mellitus. In this study, we have investigated the effect of mutation on this highly conserved residue on a KirBac channel (prokaryotic homolog of mammalian Kir6.2). We provide the crystal structure of the mutant KirBac3.1 W46R (equivalent to W68R in Kir6.2) and its conformational flexibility properties using HDX-MS. In addition, the detailed dynamical view of the mutant during the gating was investigated using the in silico method. Finally, functional assays have been performed. A comparison of important structural determinants for the gating mechanism between the wild type KirBac and the mutant W46R suggests interesting structural and dynamical clues and a mechanism of action of the mutation that leads to the gain of function.

New Structural insights into Kir channel gating from molecular simulations, HDX-MS and functional studies.

Inward rectifier potassium (Kir) channels play diverse and important roles in shaping action potentials in biological membranes. An increasing number of diseases are now known to be directly associated with abnormal Kir function. However, the gating of Kir still remains unknown. To increase our understanding of its gating mechanism, a dynamical view of the entire channel is essential. Here the gating activation was studied using a recent developped in silico method, MDeNM, which combines normal mode analysis and molecular dynamics simulations that showed for the very first time the importance of interrelated collective and localized conformational movements. In particular, we highlighted the role played by concerted movements of the different regions throughout the entire protein, such as the cytoplasmic and transmembrane domains and the slide helices. In addition, the HDX-MS analysis achieved in these studies provided a comprehensive and detailed view of the dynamics associated with open/closed transition of the Kir channel in coherence with the theoretical results. MDeNM gives access to the probability of the different opening states that are in agreement with our electrophysiological experiments. The investigations presented in this article are important to remedy dysfunctional channels and are of interest for designing new pharmacological compounds.

Architecture of TAF11/TAF13/TBP complex suggests novel regulation properties of general transcription factor TFIID.

General transcription factor TFIID is a key component of RNA polymerase II transcription initiation. Human TFIID is a megadalton-sized complex comprising TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). TBP binds to core promoter DNA, recognizing the TATA-box. We identified a ternary complex formed by TBP and the histone fold (HF) domain-containing TFIID subunits TAF11 and TAF13. We demonstrate that TAF11/TAF13 competes for TBP binding with TATA-box DNA, and also with the N-terminal domain of TAF1 previously implicated in TATA-box mimicry. In an integrative approach combining crystal coordinates, biochemical analyses and data from cross-linking mass-spectrometry (CLMS), we determine the architecture of the TAF11/TAF13/TBP complex, revealing TAF11/TAF13 interaction with the DNA binding surface of TBP. We identify a highly conserved C-terminal TBP-interaction domain (CTID) in TAF13, which is essential for supporting cell growth. Our results thus have implications for cellular TFIID assembly and suggest a novel regulatory state for TFIID function.

The vaccinia virus DNA polymerase structure provides insights into the mode of processivity factor binding.

Vaccinia virus (VACV), the prototype member of the Poxviridae, replicates in the cytoplasm of an infected cell. The catalytic subunit of the DNA polymerase E9 binds the heterodimeric processivity factor A20/D4 to form the functional polymerase holoenzyme. Here we present the crystal structure of full-length E9 at 2.7 Å resolution that permits identification of important poxvirus-specific structural insertions. One insertion in the palm domain interacts with C-terminal residues of A20 and thus serves as the processivity factor-binding site. This is in strong contrast to all other family B polymerases that bind their co-factors at the C terminus of the thumb domain. The VACV E9 structure also permits rationalization of polymerase inhibitor resistance mutations when compared with the closely related eukaryotic polymerase delta-DNA complex.

Dynamic distinctions in the Na+/Ca2+ exchanger adopting the inward- and outward-facing conformational states.

Na+/Ca2+ exchanger (NCX) proteins operate through the alternating access mechanism, where the ion-binding pocket is exposed in succession either to the extracellular or the intracellular face of the membrane. The archaeal NCX_Mj (Methanococcus jannaschii NCX) system was used to resolve the backbone dynamics in the inward-facing (IF) and outward-facing (OF) states by analyzing purified preparations of apo- and ion-bound forms of NCX_Mj-WT and its mutant, NCX_Mj-5L6-8. First, the exposure of extracellular and cytosolic vestibules to the bulk phase was evaluated as the reactivity of single cysteine mutants to a fluorescent probe, verifying that NCX_Mj-WT and NCX_Mj-5L6-8 preferentially adopt the OF and IF states, respectively. Next, hydrogen-deuterium exchange-mass spectrometry (HDX-MS) was employed to analyze the backbone dynamics profiles in proteins, preferentially adopting the OF (WT) and IF (5L6-8) states either in the presence or absence of ions. Characteristic differences in the backbone dynamics were identified between apo NCX_Mj-WT and NCX_Mj-5L6-8, thereby underscoring specific conformational patterns owned by the OF and IF states. Saturating concentrations of Na+ or Ca2+ specifically modify HDX patterns, revealing that the ion-bound/occluded states are much more stable (rigid) in the OF than in the IF state. Conformational differences observed in the ion-occluded OF and IF states can account for diversifying the ion-release dynamics and apparent affinity (Km ) at opposite sides of the membrane, where specific structure-dynamic elements can effectively match the rates of bidirectional ion movements at physiological ion concentrations.

Conformational Dynamics and Interactions of Membrane Proteins by Hydrogen/Deuterium Mass Spectrometry.

Hydrogen/deuterium exchange associated with mass spectrometry has been recently used to characterize the dynamics and the interactions of membrane proteins. Here we describe experimental workflow enabling localization of the regions involved in conformational changes or interactions.

Asymmetric Preorganization of Inverted Pair Residues in the Sodium-Calcium Exchanger.

In analogy with many other proteins, Na(+)/Ca(2+) exchangers (NCX) adapt an inverted twofold symmetry of repeated structural elements, while exhibiting a functional asymmetry by stabilizing an outward-facing conformation. Here, structure-based mutant analyses of the Methanococcus jannaschii Na(+)/Ca(2+) exchanger (NCX_Mj) were performed in conjunction with HDX-MS (hydrogen/deuterium exchange mass spectrometry) to identify the structure-dynamic determinants of functional asymmetry. HDX-MS identified hallmark differences in backbone dynamics at ion-coordinating residues of apo-NCX_Mj, whereas Na(+)or Ca(2+) binding to the respective sites induced relatively small, but specific, changes in backbone dynamics. Mutant analysis identified ion-coordinating residues affecting the catalytic capacity (kcat/Km), but not the stability of the outward-facing conformation. In contrast, distinct "noncatalytic" residues (adjacent to the ion-coordinating residues) control the stability of the outward-facing conformation, but not the catalytic capacity. The helix-breaking signature sequences (GTSLPE) on the α1 and α2 repeats (at the ion-binding core) differ in their folding/unfolding dynamics, while providing asymmetric contributions to transport activities. The present data strongly support the idea that asymmetric preorganization of the ligand-free ion-pocket predefines catalytic reorganization of ion-bound residues, where secondary interactions with adjacent residues couple the alternating access. These findings provide a structure-dynamic basis for ion-coupled alternating access in NCX and similar proteins.

Mapping the Binding Site of a Cross-Reactive Plasmodium falciparum PfEMP1 Monoclonal Antibody Inhibitory of ICAM-1 Binding.

The virulence of Plasmodium falciparum is linked to the ability of infected erythrocytes (IE) to adhere to the vascular endothelium, mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1). In this article, we report the functional characterization of an mAb that recognizes a panel of PfEMP1s and inhibits ICAM-1 binding. The 24E9 mouse mAb was raised against PFD1235w DBLß3_D4, a domain from the group A PfEMP1s associated with severe malaria. 24E9 recognizes native PfEMP1 expressed on the IE surface and shows cross-reactivity with and cross-inhibition of the ICAM-1 binding capacity of domain cassette 4 PfEMP1s. 24E9 Fab fragments bind DBLß3_D4 with nanomolar affinity and inhibit ICAM-1 binding of domain cassette 4-expressing IE. The antigenic regions targeted by 24E9 Fab were identified by hydrogen/deuterium exchange mass spectrometry and revealed three discrete peptides that are solvent protected in the complex. When mapped onto a homology model of DBLß3_D4, these cluster to a defined, surface-exposed region on the convex surface of DBLß3_D4. Mutagenesis confirmed that the site most strongly protected is necessary for 24E9 binding, which is consistent with a low-resolution structure of the DBLß3_D4::24E9 Fab complex derived from small-angle x-ray scattering. The convex surface of DBLß3_D4 has previously been shown to contain the ICAM-1 binding site of DBLß domains, suggesting that the mAb acts by occluding the ICAM-1 binding surface. Conserved epitopes, such as those targeted by 24E9, are promising candidates for the inclusion in a vaccine interfering with ICAM-1-specific adhesion of group A PfEMP1 expressed by P. falciparum IE during severe malaria.

Cloning of the first human anti-JCPyV/VP1 neutralizing monoclonal antibody: epitope definition and implications in risk stratification of patients under natalizumab therapy.

JC virus (JCPyV) has gained novel clinical importance as cause of progressive multifocal leukoencephalopathy (PML), a rare demyelinating disease recently associated to immunomodulatory drugs, such as natalizumab used in multiple sclerosis (MS) cases. Little is known about the mechanisms leading to PML, and this makes the need of PML risk stratification among natalizumab-treated patients very compelling. Clinical and laboratory-based risk-stratification markers have been proposed, one of these is represented by the JCPyV-seropositive status, which includes about 54% of MS patients. We recently proposed to investigate the possible protective role of neutralizing humoral immune response in preventing JCPyV reactivation. In this proof-of-concept study, by cloning the first human monoclonal antibody (GRE1) directed against a neutralizing epitope on JCPyV/VP1, we optimized a robust anti-JCPyV neutralization assay. This allowed us to evaluate the neutralizing activity in JCPyV-positive sera from MS patients, demonstrating the lack of correlation between the level of anti-JCPyV antibody and anti-JCPyV neutralizing activity. Relevant consequences may derive from future clinical studies induced by these findings; indeed the study of the serum anti-JCPyV neutralizing activity could allow not only a better risk stratification of the patients during natalizumab treatment, but also a better understanding of the pathophysiological mechanisms leading to PML, highlighting the contribution of peripheral versus central nervous system JCPyV reactivation. Noteworthy, the availability of GRE1 could allow the design of novel immunoprophylactic strategies during the immunomodulatory treatment.

Exploring the conformational dynamics of the bovine ADP/ATP carrier in mitochondria.

The mitochondrial ADP/ATP carrier catalyzes the transport of ADP and ATP across the mitochondrial inner membrane by switching between two different conformations. They can be blocked by two inhibitors: carboxyatractyloside (CATR) and bongkrekic acid (BA). Our understanding of the ADP/ATP transport process is largely based on analysis of structural differences between the individual inhibited states. The X-ray crystallographic three-dimensional structure of bovine ADP/ATP carrier isoform 1 (bAnc1p) complexed with CATR was determined, but the structure of the BA-carrier complex remains unknown. We recently investigated the conformational dynamics of bAnc1p in detergent solution using hydrogen/deuterium exchange and mass spectrometry (HDX-MS). This study shed light on some features of ADP/ATP translocation, but the mechanism itself and the organization of bAnc1p in the membrane required further investigation. This paper describes the first study of bAnc1p in the mitochondria on the whole-protein scale using HDX-MS. Membrane-embedded bAnc1p was deuterated and purified under HDX-MS-compatible conditions. Our results for the carrier in the mitochondrial inner membrane differed from those published for the carrier in a detergent solution. These differences were mainly in the upper half of the cavity that globally showed a limited H/D exchange whatever the complex analyzed and at the level of the matrix loops that were less accessible to the solvent in the BA-carrier complex than in the CATR-carrier complex. They are discussed with respect to published data for bAnc1p and have provided new insights into the conformation of the matrix loops of the bovine carrier in complex with BA in mitochondria.

Dynamics of a bacterial multidrug ABC transporter in the inward- and outward-facing conformations.

The study of membrane proteins remains a challenging task, and approaches to unravel their dynamics are scarce. Here, we applied hydrogen/deuterium exchange (HDX) coupled to mass spectrometry to probe the motions of a bacterial multidrug ATP-binding cassette (ABC) transporter, BmrA, in the inward-facing (resting state) and outward-facing (ATP-bound) conformations. Trypsin digestion and global or local HDX support the transition between inward- and outward-facing conformations during the catalytic cycle of BmrA. However, in the resting state, peptides from the two intracellular domains, especially ICD2, show a much faster HDX than in the closed state. This shows that these two subdomains are very flexible in this conformation. Additionally, molecular dynamics simulations suggest a large fluctuation of the Cα positions from ICD2 residues in the inward-facing conformation of a related transporter, MsbA. These results highlight the unexpected flexibility of ABC exporters in the resting state and underline the power of HDX coupled to mass spectrometry to explore conformational changes and dynamics of large membrane proteins.

Accessibility changes within diphtheria toxin T domain upon membrane penetration probed by hydrogen exchange and mass spectrometry.

The translocation domain of diphtheria toxin inserts in membrane and becomes functional when the pH inside endosomes is acid. At that stage, the domain is in a partially folded state; this prevents the use of high-resolution methods for the characterization of its functional structure. On that purpose, we report here the use of hydrogen/deuterium exchange experiments coupled to mass spectrometry. The conformation changes during the different steps of insertion into lipid bilayer are monitored with a resolution of few residues. Three parts of the translocation domain can be distinguished. With a high protection against exchange, the C-terminal hydrophobic helical hairpin is embedded in the membrane. Despite a lower protection, a significant effect in the presence of lipid vesicles shows that the N-terminal part is in interaction with the membrane interface. The sensitivity to the ionic strength indicates that electrostatic interactions are important for the binding. The middle part of the domain has an intermediate protection; this suggests that this part of the domain can be embedded within the membrane but remains quite dynamic. These results provide unprecedented insight into the structure reorganization of the protein to go from a soluble state to a membrane-inserted one.

Yeast ADP/ATP carrier isoform 2: conformational dynamics and role of the RRRMMM signature sequence methionines.

The mitochondrial ADP/ATP carrier, or Ancp, is a member of the mitochondrial carrier family responsible for exchanging ADP and ATP across the mitochondrial inner membrane. ADP/ATP transport involves Ancp switching between two conformational states. These can be analyzed using specific inhibitors, carboxyatractyloside (CATR) and bongkrekic acid (BA). The high resolution three-dimensional structure of bovine Anc1p (bAnc1p), as a CATR-carrier complex, has been solved. However, because the structure of the BA-carrier complex has not yet been determined, the detailed mechanism of transport remains unknown. Recently, sample processing for hydrogen/deuterium exchange experiments coupled to mass spectrometry was improved, providing novel insights into bAnc1p conformational transitions due to inhibitor binding. In this work we performed both hydrogen/deuterium exchange-mass spectrometry experiments and genetic manipulations. Because these are very difficult to apply with bovine Anc1p, we used Saccharomyces cerevisiae Anc isoform 2 (ScAnc2p). Significant differences in solvent accessibility were observed throughout the amino acid sequence for ScAnc2p complexed to either CATR or BA. Interestingly, in detergent solution, the conformational dynamics of ScAnc2p were dissimilar to those of bAnc1p, in particular for the upper half of the cavity, toward the intermembrane space, and the m2 loop, which is thought to be easily accessible to the solvent from the matrix in bAnc1p. Our study then focused on the methionyl residues of the Ancp signature sequence, RRRMMM. All our results indicate that the methionine cluster is involved in the ADP/ATP transport mechanism and confirm that the Ancp cavity is a highly dynamic structure.

NADPH oxidase (NOX) isoforms are inhibited by celastrol with a dual mode of action.

BACKGROUND: Celastrol is one of several bioactive compounds extracted from the medicinal plant Tripterygium wilfordii. Celastrol is used to treat inflammatory conditions, and shows benefits in models of neurodegenerative disease, cancer and arthritis, although its mechanism of action is incompletely understood. EXPERIMENTAL APPROACH: Celastrol was tested on human NADPH oxidases (NOXs) using a panel of experiments: production of reactive oxygen species and oxygen consumption by NOX enzymes, xanthine oxidase activity, cell toxicity, phagocyte oxidase subunit translocation, and binding to cytosolic subunits of NOX enzymes. The effect of celastrol was compared with diphenyleneiodonium, an established inhibitor of flavoproteins. KEY RESULTS: Low concentrations of celastrol completely inhibited NOX1, NOX2, NOX4 and NOX5 within minutes with concentration-response curves exhibiting higher Hill coefficients and lower IC50 values for NOX1 and NOX2 compared with NOX4 and NOX5, suggesting differences in their mode of action. In a cell-free system, celastrol had an IC50 of 1.24 and 8.4 µM for NOX2 and NOX5, respectively. Cytotoxicity, oxidant scavenging, and inhibition of p47(phox) translocation could not account for NOX inhibition. Celastrol bound to a recombinant p47(phox) and disrupted the binding of the proline rich region of p22(phox) to the tandem SH3 domain of p47(phox) and NOXO1, the cytosolic subunits of NOX2 and NOX1, respectively. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that celastrol is a potent inhibitor of NOX enzymes in general with increased potency against NOX1 and NOX2. Furthermore, inhibition of NOX1 and NOX2 was mediated via a novel mode of action, namely inhibition of a functional association between cytosolic subunits and the membrane flavocytochrome.

Solid-Phase Cross-Linking (SPCL): a new tool for protein structure studies.

A wide range of chemical reagents are available to study the protein-protein interactions or protein structures. After reaction with such chemicals, covalently modified proteins are digested, resulting in shorter peptides that are analyzed by mass spectrometry (MS). Used especially when NMR of X-ray data are lacking, this methodology requires the identification of modified species carrying relevant information, among the unmodified peptides. To overcome the drawbacks of existing methods, we propose a more direct strategy relying on the synthesis of solid-supported cleavable monofunctional reagents and cross-linkers that react with proteins and that selectively release, after protein digestion and washings, the modified peptide fragments ready for MS analysis. Using this Solid-Phase Cross-Linking (SPCL) strategy, only modified sequences are analyzed and consistent data can be easily obtained since the signals of interest are not masked or suppressed by over-represented unmodified materials.

A new CZE method for profiling human serum albumin and its related forms to assess the quality of biopharmaceuticals.

We present a new CZE method, which uses a polyethylene oxide-coated capillary to separate native HSA from more than five of its structural variants. These variants include oxidized, truncated, and cysteinylated forms of HSA which can all be found in biopharmaceutical products. Both CE and MS confirmed the high degree of heterogeneity of HSA preparations. Recovery studies demonstrated that adsorption of HSA on the capillary was significantly reduced under the conditions we developed, which led to a satisfactory repeatability (RSD for migration times and relative peak areas were less than 0.2 and 7.0%, respectively). Assignment of the main peaks was attempted using in vitro degraded/stressed HSA. We used our method to test batch-to-batch comparability and detected slight quantitative differences in the proportion of native HSA in batches produced from different fractionation methods.

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.

p47phox molecular activation for assembly of the neutrophil NADPH oxidase complex.

The p47(phox) cytosolic factor from neutrophilic NADPH oxidase has always been resistant to crystallogenesis trials due to its modular organization leading to relative flexibility. Hydrogen/deuterium exchange coupled to mass spectrometry was used to obtain structural information on the conformational mechanism that underlies p47(phox) activation. We confirmed a relative opening of the protein with exposure of the SH3 Src loops that are known to bind p22(phox) upon activation. A new surface was shown to be unmasked after activation, representing a potential autoinhibitory surface that may block the interaction of the PX domain with the membrane in the resting state. Within this surface, we identified 2 residues involved in the interaction with the PX domain. The double mutant R162A/D166A showed a higher affinity for specific phospholipids but none for the C-terminal part of p22(phox), reflecting an intermediate conformation between the autoinhibited and activated forms.

Effective removal of nonionic detergents in protein mass spectrometry, hydrogen/deuterium exchange, and proteomics.

Detergents are frequently used for protein isolation and solubilization. Their presence is crucial in membrane protein protocols or in lipid raft proteomics. However, they are usually poorly compatible with mass spectrometry. Several different sample preparation protocols are routinely used, but they are either laborious or suffer from sample losses. Here, we describe our alternative method for nonionic detergent removal. It is based on selective detergent extraction after capture of the sample on a reversed phase cartridge. The extraction is performed by chlorinated solvents and works well for polyoxyethylene based nonionic detergents, but also for polymers like polyethylene and propylene glycol. Detergent removal can be also carried out on the protein level but a special care must be taken with hydrophobic proteins. In such cases, it is preferable to perform detergent removal after proteolysis which digests the protein to peptides and reduces the hydrophobicity. The method can easily be automated and is compatible with hydrogen/deuterium exchange coupled to mass spectrometry.