2'-deoxy-ADPR activates human TRPM2 faster than ADPR and thereby induces higher currents at physiological Ca2+ concentrations.

TRPM2 is a Ca2+ permeable, non-selective cation channel in the plasma membrane that is involved in the innate immune response regulating, for example, chemotaxis in neutrophils and cytokine secretion in monocytes and macrophages. The intracellular adenine nucleotides ADP-ribose (ADPR) and 2'-deoxy-ADPR (2dADPR) activate the channel, in combination with their co-agonist Ca2+. Interestingly, activation of human TRPM2 (hsTRPM2) by 2dADPR is much more effective than activation by ADPR. However, the underlying mechanism of the nucleotides' differential effect on the channel is not yet fully understood. In this study, we performed whole-cell patch clamp experiments with HEK293 cells heterologously expressing hsTRPM2. We show that 2dADPR has an approx. 4-fold higher Ca2+ sensitivity than ADPR (EC50 = 190 and 690 nM). This allows 2dADPR to activate the channel at lower and thus physiological intracellular Ca2+ concentrations. Kinetic analysis of our data reveals that activation by 2dADPR is faster than activation by ADPR. Mutation in a calmodulin binding N-terminal IQ-like motif in hsTRPM2 completely abrogated channel activation by both agonists. However, mutation of a single amino acid residue (W1355A) in the C-terminus of hsTRPM2, at a site of extensive inter-domain interaction, resulted in slower activation by 2dADPR and neutralized the difference in rate of activation between the two agonists. Taken together, we propose a mechanism by which 2dADPR induces higher hsTRPM2 currents than ADPR by means of faster channel activation. The finding that 2dADPR has a higher Ca2+ sensitivity than ADPR may indicate that 2dADPR rather than ADPR activates hsTRPM2 in physiological contexts such as the innate immune response.

Mice heterozygous for an osteogenesis imperfecta-linked MBTPS2 variant display a compromised subchondral osteocyte lacunocanalicular network associated with abnormal articular cartilage.

Missense variants in the MBTPS2 gene, located on the X chromosome, have been associated with an X-linked recessive form of osteogenesis imperfecta (X-OI), an inherited bone dysplasia characterized by multiple and recurrent bone fractures, short stature, and various skeletal deformities in affected individuals. The role of site-2 protease, encoded by MBTPS2, and the molecular pathomechanism underlying the disease are to date elusive. This study is the first to report on the generation of two Mbtps2 mouse models, a knock-in mouse carrying one of the disease-causative MBTPS2 variants (N455S) and a Mbtps2 knock-out (ko) mouse. Because both loss-of-function variants lead to embryonic lethality in hemizygous male mutant mice, we performed a comprehensive skeletal analysis of heterozygous Mbtps2+/N455S and Mbtps2+/ko female mice. Both models displayed osteochondral abnormalities such as thinned subchondral bone, altered subchondral osteocyte interconnectivity as well as thickened articular cartilage with chondrocyte clustering, altogether resembling an early osteoarthritis (OA) phenotype. However, distant from the joints, no alterations in the bone mass and turnover could be detected in either of the mutant mice. Based on our findings we conclude that MBTPS2 haploinsufficiency results in early OA-like alterations in the articular cartilage and underlying subchondral bone, which likely precede the development of typical OI phenotype in bone. Our study provides first evidence for a potential role of site-2 protease for maintaining homeostasis of both bone and cartilage.

Interactions of TonB-dependent transporter FoxA with siderophores and antibiotics that affect binding, uptake, and signal transduction.

The outer membrane of gram-negative bacteria prevents many antibiotics from reaching intracellular targets. However, some antimicrobials can take advantage of iron import transporters to cross this barrier. We showed previously that the thiopeptide antibiotic thiocillin exploits the nocardamine xenosiderophore transporter, FoxA, of the opportunistic pathogen Pseudomonas aeruginosa for uptake. Here, we show that FoxA also transports the xenosiderophore bisucaberin and describe at 2.5 Å resolution the crystal structure of bisucaberin bound to FoxA. Bisucaberin is distinct from other siderophores because it forms a 3:2 rather than 1:1 siderophore-iron complex. Mutations in a single extracellular loop of FoxA differentially affected nocardamine, thiocillin, and bisucaberin binding, uptake, and signal transduction. These results show that in addition to modulating ligand binding, the extracellular loops of siderophore transporters are of fundamental importance for controlling ligand uptake and its regulatory consequences, which have implications for the development of siderophore-antibiotic conjugates to treat difficult infections.

Stopped-flow-time-resolved SAXS for studies of ligand-driven protein dimerization.

Protein function is highly dependent on conformational changes and association or dissociation into numerous oligomeric states. Stopped-flow approaches are suitable for probing transient kinetics in proteins, and combining this approach with small-angle X-ray scattering offers an excellent probe into the structural kinetics of protein function. In this chapter we describe in detail the methodological aspects of our recent investigation of ATP-driven dimerization of nucleotide-binding domains from the bacterial transporter MsbA using stopped-flow small-angle X-ray scattering experiments. Despite extensive studies into the structure and function of MsbA, the structural-temporal insights into the conformational rearrangements and transient intermediates along the pathway in this transporter are missing. In our stopped-flow experiments we observe the rapid formation of a transient protein dimer and subsequent dimer decay over hundreds of seconds. Thus, this approach can be used to detect kinetic parameters associated with conformational changes over a wide range of time-scales for soluble and membrane proteins.

Studying integral membrane protein by SANS using stealth reconstitution systems.

Structural studies of integral membrane proteins (IMPs) are challenging as many of them require a lipid environment for full activity and stability. Reconstitution of IMPs into carrier systems such as nanodiscs or Salipro that mimic the native lipidic environment allow structural studies of membrane proteins in solution. The difficulty with this approach when applied to scattering techniques is the contribution of the carrier system to the scattering intensity and the subsequent challenging data analysis. Recently, so-called stealth carrier systems have been developed and applied to small-angle neutron scattering (SANS) studies of integral membrane proteins that become invisible to neutrons due to specific deuteration and solvent contrast-variation. In this chapter, we describe in detail how the well-studied ATP-binding cassette (ABC) transporter protein MsbA can be reconstituted into stealth nanodiscs and subsequently be studied by SANS. This approach allows for a direct observation of the scattering signal from MsbA without the contribution of the surrounding carrier system and enables detection of different conformational states. The protocols can also be adapted to other stealth carrier systems (such as stealth Salipro).

New insights into P2X7 receptor regulation: Ca2+-calmodulin and GDP bind to the soluble P2X7 ballast domain.

P2X7 receptors are nonselective cation channels that are activated by extracellular ATP and play important roles in inflammation. They differ from other P2X family members by a large intracellular C-terminus that mediates diverse signaling processes that are little understood. A recent cryo-EM study revealed that the C-terminus of the P2X7 receptor forms a unique cytoplasmic ballast domain that possesses a GDP-binding site as well as a dinuclear Zn2+ site. However, the molecular basis for the regulatory function of the ballast domain as well as the interplay between the various ligands remain unclear. Here, we successfully expressed a soluble trimeric P2X7 ballast domain (P2X7BD) and characterized its ligand binding properties using a biophysical approach. We identified calmodulin (CaM)-binding regions within the ballast domain and found that binding of Ca2+-CaM and GDP to P2X7BD have opposite effects on its stability. Small-angle X-ray scattering experiments indicate that Ca2+-CaM binding disrupts the trimeric state of P2X7BD. Our results provide a possible framework for the intracellular regulation of the P2X7 receptor.

The crystal structure of TRPM2 MHR1/2 domain reveals a conserved Zn2+ -binding domain essential for structural integrity and channel activity.

Transient receptor potential melastatin 2 (TRPM2) is a Ca2+ -permeable, nonselective cation channel involved in diverse physiological processes such as immune response, apoptosis, and body temperature sensing. TRPM2 is activated by ADP-ribose (ADPR) and 2'-deoxy-ADPR in a Ca2+ -dependent manner. While two distinct binding sites exist for ADPR that exert different functions dependent on the species, the involvement of either binding site regarding the superagonistic effect of 2'-deoxy-ADPR is not clear yet. Here, we report the crystal structure of the MHR1/2 domain of TRPM2 from zebrafish (Danio rerio), and show that both ligands bind to this domain and activate the channel. We identified a so far unrecognized Zn2+ -binding domain that was not resolved in previous cryo-EM structures and that is conserved in most TRPM channels. In combination with patch clamp experiments we comprehensively characterize the effect of the Zn2+ -binding domain on TRPM2 activation. Our results provide insight into a conserved motif essential for structural integrity and channel activity.

cADPR Does Not Activate TRPM2.

cADPR is a second messenger that releases Ca2+ from intracellular stores via the ryanodine receptor. Over more than 15 years, it has been controversially discussed whether cADPR also contributes to the activation of the nucleotide-gated cation channel TRPM2. While some groups have observed activation of TRPM2 by cADPR alone or in synergy with ADPR, sometimes only at 37 °C, others have argued that this is due to the contamination of cADPR by ADPR. The identification of a novel nucleotide-binding site in the N-terminus of TRPM2 that binds ADPR in a horseshoe-like conformation resembling cADPR as well as the cADPR antagonist 8-Br-cADPR, and another report that demonstrates activation of TRPM2 by binding of cADPR to the NUDT9H domain raised the question again and led us to revisit the topic. Here we show that (i) the N-terminal MHR1/2 domain and the C-terminal NUDT9H domain are required for activation of human TRPM2 by ADPR and 2'-deoxy-ADPR (2dADPR), (ii) that pure cADPR does not activate TRPM2 under a variety of conditions that have previously been shown to result in channel activation, (iii) the cADPR antagonist 8-Br-cADPR also inhibits activation of TRPM2 by ADPR, and (iv) cADPR does not bind to the MHR1/2 domain of TRPM2 while ADPR does.

Population Dynamics of Stretching Excitations of p-Azido-phenylalanine Incorporated in Calmodulin-Peptide Complexes.

We genetically incorporated the unnatural amino acid p-azido-phenylalanine (AzF) into the ubiquitous Ca2+ sensor protein calmodulin (CaM) in complex with different peptides to explore the response of the azido stretching line shape to varying binding motifs with femtosecond infrared spectroscopy. The dynamic response of the azido stretching mode varies in different CaM-peptide complexes. We model these dynamics as coherent excitations of Fermi resonances and extract a lifetime of the azido stretching vibration of about 1 ps. The resulting model parameters are commensurate with the linear infrared absorption lineshapes which suggests that the conformation-sensitive vibrational lineshape could be composed of Fermi resonances that differ between the protein-peptide complexes.

Cryo-EM structure of MsbA in saposin-lipid nanoparticles (Salipro) provides insights into nucleotide coordination.

The ATP-binding cassette transporter MsbA is a lipid flippase, translocating lipid A, glycolipids, and lipopolysaccharides from the inner to the outer leaflet of the inner membrane of Gram-negative bacteria. It has been used as a model system for time-resolved structural studies as several MsbA structures in different states and reconstitution systems (detergent/nanodiscs/peptidiscs) are available. However, due to the limited resolution of the available structures, detailed structural information on the bound nucleotides has remained elusive. Here, we have reconstituted MsbA in saposin A-lipoprotein nanoparticles (Salipro) and determined the structure of ADP-vanadate-bound MsbA by single-particle cryo-electron microscopy to 3.5 Å resolution. This procedure has resulted in significantly improved resolution and enabled us to model all side chains and visualise detailed ADP-vanadate interactions in the nucleotide-binding domains. The approach may be applicable to other dynamic membrane proteins.

Computational models in the service of X-ray and cryo-electron microscopy structure determination.

Critical assessment of structure prediction (CASP) conducts community experiments to determine the state of the art in computing protein structure from amino acid sequence. The process relies on the experimental community providing information about not yet public or about to be solved structures, for use as targets. For some targets, the experimental structure is not solved in time for use in CASP. Calculated structure accuracy improved dramatically in this round, implying that models should now be much more useful for resolving many sorts of experimental difficulties. To test this, selected models for seven unsolved targets were provided to the experimental groups. These models were from the AlphaFold2 group, who overall submitted the most accurate predictions in CASP14. Four targets were solved with the aid of the models, and, additionally, the structure of an already solved target was improved. An a posteriori analysis showed that, in some cases, models from other groups would also be effective. This paper provides accounts of the successful application of models to structure determination, including molecular replacement for X-ray crystallography, backbone tracing and sequence positioning in a cryo-electron microscopy structure, and correction of local features. The results suggest that, in future, there will be greatly increased synergy between computational and experimental approaches to structure determination.

Structural insights into a novel family of integral membrane siderophore reductases.

Gram-negative bacteria take up the essential ion Fe3+ as ferric-siderophore complexes through their outer membrane using TonB-dependent transporters. However, the subsequent route through the inner membrane differs across many bacterial species and siderophore chemistries and is not understood in detail. Here, we report the crystal structure of the inner membrane protein FoxB (from Pseudomonas aeruginosa) that is involved in Fe-siderophore uptake. The structure revealed a fold with two tightly bound heme molecules. In combination with in vitro reduction assays and in vivo iron uptake studies, these results establish FoxB as an inner membrane reductase involved in the release of iron from ferrioxamine during Fe-siderophore uptake.

Analysis of ligand binding and resulting conformational changes in pyrophosphatase NUDT9.

Nudix hydrolase 9 (NUDT9) is a member of the nucleoside linked to another moiety X (NUDIX) protein superfamily, which hydrolyses a broad spectrum of organic pyrophosphates from metabolic processes. ADP-ribose (ADPR) has been the only known endogenous substrate accepted by NUDT9 so far. The Ca2+ -permeable transient receptor potential melastatin subfamily 2 (TRPM2) channel contains a homologous NUDT9-homology (NUDT9H) domain and is activated by ADPR. Sustained Ca2+ influx via ADPR-activated TRPM2 triggers apoptotic mechanisms. Thus, a precise regulation of cellular ADPR levels by NUDT9 is essential. A detailed characterization of the enzyme-substrate interaction would help to understand the high substrate specificity of NUDT9. Here, we analysed ligand binding to NUDT9 using a variety of biophysical techniques. We identified 2'-deoxy-ADPR as an additional substrate for NUDT9. Similar enzyme kinetics and binding affinities were determined for the two ligands. The high-affinity binding was preserved in NUDT9 containing the mutated NUDIX box derived from the human NUDT9H domain. NMR spectroscopy indicated that ADPR and 2'-deoxy-ADPR bind to the same binding site of NUDT9. Backbone resonance assignment and subsequent molecular docking allowed further characterization of the binding pocket. Substantial conformational changes of NUDT9 upon ligand binding were observed which might allow for the development of NUDT9-based ADPR fluorescence resonance energy transfer sensors that may help with the analysis of ADPR signalling processes in cells in the future.

FoldAffinity: binding affinities from nDSF experiments.

Differential scanning fluorimetry (DSF) using the inherent fluorescence of proteins (nDSF) is a popular technique to evaluate thermal protein stability in different conditions (e.g. buffer, pH). In many cases, ligand binding increases thermal stability of a protein and often this can be detected as a clear shift in nDSF experiments. Here, we evaluate binding affinity quantification based on thermal shifts. We present four protein systems with different binding affinity ligands, ranging from nM to high µM. Our study suggests that binding affinities determined by isothermal analysis are in better agreement with those from established biophysical techniques (ITC and MST) compared to apparent Kds obtained from melting temperatures. In addition, we describe a method to optionally fit the heat capacity change upon unfolding ([Formula: see text]) during the isothermal analysis. This publication includes the release of a web server for easy and accessible application of isothermal analysis to nDSF data.

Nocardamine-Dependent Iron Uptake in Pseudomonas aeruginosa: Exclusive Involvement of the FoxA Outer Membrane Transporter.

Iron is a key nutrient for almost all living organisms. Paradoxically, it is poorly soluble and consequently poorly bioavailable. Bacteria have thus developed multiple strategies to access this metal. One of the most common consists of the use of siderophores, small compounds that chelate ferric iron with very high affinity. Many bacteria are able to produce their own siderophores or use those produced by other microorganisms (exosiderophores) in a piracy strategy. Pseudomonas aeruginosa produces two siderophores, pyoverdine and pyochelin, and is also able to use a large panel of exosiderophores. We investigated the ability of P. aeruginosa to use nocardamine (NOCA) and ferrioxamine B (DFOB) as exosiderophores under iron-limited planktonic growth conditions. Proteomic and RT-qPCR approaches showed induction of the transcription and expression of the outer membrane transporter FoxA in the presence of NOCA or DFOB in the bacterial environment. Expression of the proteins of the heme- or pyoverdine- and pyochelin-dependent iron uptake pathways was not affected by the presence of these two tris-hydroxamate siderophores. 55Fe uptake assays using foxA mutants showed ferri-NOCA to be exclusively transported by FoxA, whereas ferri-DFOB was transported by FoxA and at least one other unidentified transporter. The crystal structure of FoxA complexed with NOCA-Fe revealed very similar siderophore binding sites between NOCA-Fe and DFOB-Fe. We discuss iron uptake by hydroxamate exosiderophores in P. aeruginosa cells in light of these results.

Structural Kinetics of MsbA Investigated by Stopped-Flow Time-Resolved Small-Angle X-Ray Scattering.

Recent structures of full-length ATP-binding cassette (ABC) transporter MsbA in different states indicate large conformational changes during the reaction cycle that involve transient dimerization of its nucleotide-binding domains (NBDs). However, a detailed molecular understanding of the structural changes and associated kinetics of MsbA upon ATP binding and hydrolysis is still missing. Here, we employed time-resolved small-angle X-ray scattering, initiated by stopped-flow mixing, to investigate the kinetics and accompanying structural changes of NBD dimerization (upon ATP binding) and subsequent dissociation (upon ATP hydrolysis) in the context of isolated NBDs as well as full-length MsbA in lipid nanodiscs. Our data allowed us to structurally characterize the major states involved in the process and determine time constants for NBD dimerization and dissociation. In the full-length protein, these structural transitions occur on much faster time scales, indicating close-proximity effects and structural coupling of the transmembrane domains with the NBDs.

Combined in vitro and in silico analyses of missense mutations in GNPTAB provide new insights into the molecular bases of mucolipidosis II and III alpha/beta.

Mucolipidosis (ML) II and III alpha/beta are inherited lysosomal storage disorders caused by mutations in GNPTAB encoding the α/ß-precursor of GlcNAc-1-phosphotransferase. This enzyme catalyzes the initial step in the modification of more than 70 lysosomal enzymes with mannose 6-phosphate residues to ensure their intracellular targeting to lysosomes. The so-called stealth domains in the α- and ß-subunit of GlcNAc-1-phosphotransferase were thought to be involved in substrate recognition and/or catalysis. Here, we performed in silico alignment analysis of stealth domain-containing phosphotransferases and showed that the amino acid residues Glu389 , Asp408 , His956 , and Arg986 are highly conserved between different phosphotransferases. Interestingly, mutations in these residues were identified in patients with MLII and MLIII alpha/beta. To further support the in silico findings, we also provide experimental data demonstrating that these four amino acid residues are strictly required for GlcNAc-1-phosphotransferase activity and thus may be directly involved in the enzymatic catalysis.

Soluble Regions of GlpG Influence Protein-Lipid Interactions and Lipid Distribution.

The GlpG rhomboid protease from E. coli is a well-characterized intramembrane protease that cleaves transmembrane substrates inside the lipid bilayer. Most studies have focused on the GlpG transmembrane domain containing the catalytic site, while the full-length protein, also containing a soluble cytoplasmic domain, a linker region, and a small positively charged C-terminal fragment, remains poorly understood. In this work, we used coarse-grained molecular dynamics (CGMD) simulations to investigate full-length GlpG embedded in a native-like model of the E. coli membrane. We identified differences in the distribution and clustering of phosphoglycerol(PG)-based lipids around GlpG in both leaflets depending on whether the soluble regions are present or absent. These data suggest a possible role of the cytoplasmic extensions of GlpG in the regulation of the lipid environment around GlpG, which may influence the activity of GlpG in vivo.

Ternary structure of the outer membrane transporter FoxA with resolved signalling domain provides insights into TonB-mediated siderophore uptake.

Many microbes and fungi acquire the essential ion Fe3+ through the synthesis and secretion of high-affinity chelators termed siderophores. In Gram-negative bacteria, these ferric-siderophore complexes are actively taken up using highly specific TonB-dependent transporters (TBDTs) located in the outer bacterial membrane (OM). However, the detailed mechanism of how the inner-membrane protein TonB connects to the transporters in the OM as well as the interplay between siderophore- and TonB-binding to the transporter is still poorly understood. Here, we present three crystal structures of the TBDT FoxA from Pseudomonas aeruginosa (containing a signalling domain) in complex with the siderophore ferrioxamine B and TonB and combine them with a detailed analysis of binding constants. The structures show that both siderophore and TonB-binding is required to form a translocation-competent state of the FoxA transporter in a two-step TonB-binding mechanism. The complex structure also indicates how TonB-binding influences the orientation of the signalling domain.

High-throughput stability screening for detergent-solubilized membrane proteins.

Protein stability in detergent or membrane-like environments is the bottleneck for structural studies on integral membrane proteins (IMP). Irrespective of the method to study the structure of an IMP, detergent solubilization from the membrane is usually the first step in the workflow. Here, we establish a simple, high-throughput screening method to identify optimal detergent conditions for membrane protein stabilization. We apply differential scanning fluorimetry in combination with scattering upon thermal denaturation to study the unfolding of integral membrane proteins. Nine different prokaryotic and eukaryotic membrane proteins were used as test cases to benchmark our detergent screening method. Our results show that it is possible to measure the stability and solubility of IMPs by diluting them from their initial solubilization condition into different detergents. We were able to identify groups of detergents with characteristic stabilization and destabilization effects for selected targets. We further show that fos-choline and PEG family detergents may lead to membrane protein destabilization and unfolding. Finally, we determined thenmodynamic parameters that are important indicators of IMP stability. The described protocol allows the identification of conditions that are suitable for downstream handling of membrane proteins during purification.