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The journal of biological chemistry 299(4), 104585 (2023). doi:10.1016/j.jbc.2023.104585, Emfourin (M4in) is a protein metalloprotease inhibitor recently discovered in the bacterium Serratia proteamaculans and the prototype of a new family of protein protease inhibitors with an unknown mechanism of action. Protealysin-like proteases (PLPs) of the thermolysin family are natural targets of emfourin-like inhibitors widespread in bacteria and known in archaea. The available data indicate the involvement of PLPs in interbacterial interaction as well as bacterial interaction with other organisms and likely in pathogenesis. Arguably, emfourin-like inhibitors participate in the regulation of bacterial pathogenesis by controlling PLP activity. Here, we determined the 3D structure of M4in using solution NMR spectroscopy. The obtained structure demonstrated no significant similarity to known protein structures. This structure was used to model the M4in–enzyme complex and the complex model was verified by small-angle X-ray scattering. Based on the model analysis, we propose a molecular mechanism for the inhibitor, which was confirmed by site-directed mutagenesis. We show that two spatially close flexible loop regions are critical for the inhibitor–protease interaction. One region includes aspartic acid forming a coordination bond with catalytic Zn2+ of the enzyme and the second region carries hydrophobic amino acids interacting with protease substrate binding sites. Such an active site structure corresponds to the noncanonical inhibition mechanism. This is the first demonstration of such a mechanism for protein inhibitors of thermolysin family metalloproteases, which puts forward M4in as a new basis for the development of antibacterial agents relying on selective inhibition of prominent factors of bacterial pathogenesis belonging to this family., Published by Soc., Bethesda, Md.

Alzheimer’s disease is the most widespread form of neurodegenerative disease in the world. Its clinical manifestations are explained by selective degeneration of neurons in the portions of cerebral cortex responsible for cognitive perception and memory. Amyloid peptide is accumulated beyond nerve cells at neuron contact sites into ordered strands (fibrils), forming the so-called amyloid plaques. It is found that amyloid peptide (β-amyloid or Aβ1–38–Aβ1–43), which aggregates and forms amyloid plaques in brain, is a product of successive cleavage of membrane glycoprotein-precursor of β-amyloid by β- and γ-secretases in the plasmatic membrane of neurons. The results of structural studies of this process and the main proteins involved in it are considered.

Dysregulation of post‐translational modifications (PTMs) like phosphorylation is often involved in disease. NMR may elucidate exact loci and time courses of PTMs at atomic resolution and near‐physiological conditions but requires signal assignment to individual atoms. Conventional NMR methods for this base on tedious global signal assignment that may often fail, as for large intrinsically disordered proteins (IDPs). We present a sensitive, robust alternative to rapidly obtain only the local assignment near affected signals, based on FOcused SpectroscopY (FOSY) experiments using selective polarisation transfer (SPT). We prove its efficiency by identifying two phosphorylation sites of glycogen synthase kinase 3 beta (GSK3β) in human Tau40, an IDP of 441 residues, where the extreme spectral dispersion in FOSY revealed unprimed phosphorylation also of Ser409. FOSY may broadly benefit NMR studies of PTMs and other hotspots in IDPs, including sites involved in molecular interactions., A new suite of 2D FOSY experiments focuses on one coupled nuclear spin system at a time, that is, a PTM site or a structural hotspot in general, with 3–5 known frequencies. With higher sensitivity and versatility than achievable by traditional experiments, FOSY solves the spectral dispersion problem and obtains only the relevant local NMR signal assignment in a few hours, as demonstrated for two phosphorylation sites in the 441‐residue IDP Tau.

Emfourin (M4in) from Serratia proteamaculans is a new proteinaceous inhibitor of protealysin-like proteases (PLPs), a subgroup of the well-known and widely represented metallopeptidase M4 family. Although the biological role of PLPs is debatable, data published indicate their involvement in pathogenesis, including bacterial invasion into eukaryotic cells, suppression of immune defense of some animals, and destruction of plant cell walls. Gene colocalization into a bicistronic operon observed for some PLPs and their inhibitors (as in the case of M4in) implies a mutually consistent functioning of both entities. The originality of the amino acid sequence of M4in suggests it belongs to a previously unknown protein family and this encourages structural studies. In this work, we report a near-complete assignment of 1H, 13C, and 15N resonances of recombinant M4in and its structural-dynamic properties derived from the chemical shifts. According the NMR data analysis, the M4in molecule comprises 3–5 helical elements and 4–6 β-strands, at least two of which are apparently antiparallel, ascribing this obviously globular protein to the α + β structural class. Besides, two disordered regions also exist in the central loops between the regular secondary structural elements. The obtained data provide the basis for determining the high-resolution structure as well as functioning mechanism of M4in that can be used for development of new antibacterial therapeutic strategies.

The search of a putative physiological electron acceptor for thiocyanate dehydrogenase (TcDH) newly discovered in the thiocyanate-oxidizing bacteria

The reaction between spillover hydrogen (SH) and mexidol upon high-temperature solid state catalytic isotope exchange (HSCIE) is studied experimentally and via DFT B3LYP and MP2 calculations. The distribution of isotopes and the activation energy of the reaction are determined via NMR spectro-scopy and mass spectrometry for samples of the deuterium-labeled mexidol obtained via HSCIE at 50–120°C. The DFT calculated activation energy of hydrogen transfer on the charged alumina cluster HH+(‒Al–O–)3(OH)6 is found to agree with the experimental data. A mechanism is proposed for HSCIE between the adsorbed compound and charged Bronsted acid sites formed on the surface of the support upon exposure to hydrogen spillover.

International journal of molecular sciences 23(17), 9969 (2022). doi:10.3390/ijms23179969, The search of a putative physiological electron acceptor for thiocyanate dehydrogenase (TcDH) newly discovered in the thiocyanate-oxidizing bacteria Thioalkalivibrio paradoxus revealed an unusually large, single-heme cytochrome c (CytC552), which was co-purified with TcDH from the periplasm. Recombinant CytC552, produced in Escherichia coli as a mature protein without a signal peptide, has spectral properties similar to the endogenous protein and serves as an in vitro electron acceptor in the TcDH-catalyzed reaction. The CytC552 structure determined by NMR spectroscopy reveals significant differences compared to those of the typical class I bacterial cytochromes c: a high solvent accessible surface area for the heme group and so-called “intrinsically disordered” nature of the histidine-rich N- and C-terminal regions. Comparison of the signal splitting in the heteronuclear NMR spectra of oxidized, reduced, and TcDH-bound CytC552 reveals the heme axial methionine fluxionality. The TcDH binding site on the CytC552 surface was mapped using NMR chemical shift perturbations. Putative TcDH-CytC552 complexes were reconstructed by the information-driven docking approach and used for the analysis of effective electron transfer pathways. The best pathway includes the electron hopping through His528 and Tyr164 of TcDH, and His83 of CytC552 to the heme group in accordance with pH-dependence of TcDH activity with CytC552., Published by Molecular Diversity Preservation International, Basel

Human InsR, IGF1R, and IRR receptor tyrosine kinases (RTK) of the insulin receptor subfamily play an important role in signaling pathways for a wide range of physiological processes and are directly associated with many pathologies, including neurodegenerative diseases. The disulfide-linked dimeric structure of these receptors is unique among RTKs. Sharing high sequence and structure homology, the receptors differ dramatically in their localization, expression, and functions. In this work, using high-resolution NMR spectroscopy supported by atomistic computer modeling, conformational variability of the transmembrane domains and their interactions with surrounding lipids were found to differ significantly between representatives of the subfamily. Therefore, we suggest that the heterogeneous and highly dynamic membrane environment should be taken into account in the observed diversity of the structural/dynamic organization and mechanisms of activation of InsR, IGF1R, and IRR receptors. This membrane-mediated control of receptor signaling offers an attractive prospect for the development of new targeted therapies for diseases associated with dysfunction of insulin subfamily receptors.

The crystal structure of bacterial oligopeptidase B from Serratia proteamaculans (SpOpB) in complex with a chloromethyl ketone inhibitor was determined at 2.2 Å resolution. SpOpB was crystallized in a closed (catalytically active) conformation. A single inhibitor molecule bound simultaneously to the catalytic residues S532 and H652 mimicked a tetrahedral intermediate of the catalytic reaction. A comparative analysis of the obtained structure and the structure of OpB from Trypanosoma brucei (TbOpB) in a closed conformation showed that in both enzymes, the stabilization of the D-loop (carrying the catalytic D) in a position favorable for the formation of a tetrahedral complex occurs due to interaction with the neighboring loop from the β-propeller. However, the modes of interdomain interactions were significantly different for bacterial and protozoan OpBs. Instead of a salt bridge (as in TbOpB), in SpOpB, a pair of polar residues following the catalytic D617 and a pair of neighboring arginine residues from the β-propeller domain formed complementary oppositely charged surfaces. Bioinformatics analysis and structural modeling show that all bacterial OpBs can be divided into two large groups according to these two modes of D-loop stabilization in closed conformations.

Alzheimer's disease (AD) is a severe neurodegenerative pathology with no effective treatment known. Toxic amyloid-β peptide (Aβ) oligomers play a crucial role in AD pathogenesis. All

Trichobakin (TBK) is a type-I ribosome-inactivating protein (RIP-I), acting as an extremely potent inhibitor of protein synthesis in the cell-free translation system of rabbit reticulocyte lysate (IC50: 3.5 pM). In this respect, TBK surpasses the well-studied highly homologous RIP-I trichosanthin (IC50: 20-27 pM), therefore creation of recombinant toxins based on it is of great interest. TBK needs to penetrate into cytosol through the cell membrane and specifically bind to α-sarcin/ricin loop of 28S ribosome RNA to perform the function of specific RNA depurination. At the moment, there is no detailed structural-dynamic information in solution about diverse states RIP-I can adopt at different stages on the way to protein synthesis inhibition. In this work, we report a near-complete assignment of 1H, 13C, and 15N TBK (27.3 kDa) resonances and analysis of the secondary structure based on the experimental chemical shifts data. This work will serve as a basis for further investigations of the structure, dynamics and interactions of the TBK with its molecular partners using NMR techniques.

Alzheimer's disease is an age-related pathology associated with accumulation of amyloid-β peptides, products of enzymatic cleavage of amyloid-β precursor protein (APP) by secretases. Several familial mutations causing early onset of the disease have been identified in the APP transmembrane (TM) domain. The mutations influence production of amyloid-β, but the molecular mechanisms of this effect are unclear. The "Australian" (L723P) mutation located in the C-termini of APP TM domain is associated with autosomal-dominant, early onset Alzheimer's disease. Herein, we describe the impact of familial L723P mutation on the structural-dynamic behavior of APP TM domain studied by high-resolution NMR in membrane-mimicking micelles and augmented by molecular dynamics simulations in explicit lipid bilayer. We found L723P mutation to cause local unfolding of the C-terminal turn of the APP TM domain helix and increase its accessibility to water required for cleavage of the protein backbone by γ-secretase in the ε-site, thus switching between alternative ("pathogenic" and "non-pathogenic") cleavage cascades. These findings suggest a straightforward mechanism of the pathogenesis associated with this mutation, and are of generic import for understanding the molecular-level events associated with APP sequential proteolysis resulting in accumulation of the pathogenic forms of amyloid-β. Moreover, age-related onset of Alzheimer's disease can be explained by a similar mechanism, where the effect of mutation is emulated by the impact of local environmental factors, such as oxidative stress and/or membrane lipid composition. Knowledge of the mechanisms regulating generation of amyloidogenic peptides of different lengths is essential for development of novel treatment strategies of the Alzheimer's disease.

Side chains possess a broader conformational space (compared to the backbone) and are directly affected by intra- and intermolecular interactions, hence their dynamics and the corresponding NMR relaxation data are more sensitive and informative. Nevertheless, transverse relaxation in [Formula: see text] ([Formula: see text] or [Formula: see text]) spin systems is predominantly non-measurable in uniformly [Formula: see text]-labeled proteins due to cross-correlation effects. In the present publication, we propose a number of pulse sequences for accurate and precise measurement of the dipole-dipole transverse cross-correlated relaxation rate [Formula: see text], which, similarly to [Formula: see text] measurements, provides information about the amplitudes of intramolecular dynamics. The suggested approach has allowed us to circumvent a number of obstacles that were limiting earlier applications of [Formula: see text]: (1) impossibility of transmission of the central component of the triplet of [Formula: see text] group to [Formula: see text]-acquisition via INEPT has been solved by transmission of the averaged signal of "inner" and "outer" components of the triplet; (2) direct recording of the entire triplets resulting in substantial overlap of side chain signals has been replaced by recording of individual singlets with the use of [Formula: see text]-modulated approach and constant-time evolution; (3) low sensitivity has been enhanced via proton acquisition which required special attention to a zero-quantum coherence evolution. The proposed method expands the set of "dynamics sensors" covering protein side chains and substantially improves the quality and the level of detail of experimental data describing dynamic processes in proteins and protein complexes.

The reaction of spillover hydrogen (SH) with cyclopropylglycine (cPG) in high-temperature solid-state catalytic hydrogen isotope exchange (HTCIE) has been studied experimentally and using density functional theory (DFT). NMR spectroscopy and mass spectrometry have shown a high regioselectivity and stereoselectivity for the reaction of spillover hydrogen with cPG fragments. The Gly fragment has been shown to be the most reactive in HTCIE, for which high stereoselectivity of substitution of hydrogen by deuterium has been demonstrated.

Emfourin (M4in) from Serratia proteamaculans is a new proteinaceous inhibitor of protealysin-like proteases (PLPs), a subgroup of the well-known and widely represented metallopeptidase M4 family. Although the biological role of PLPs is debatable, data published indicate their involvement in pathogenesis, including bacterial invasion into eukaryotic cells, suppression of immune defense of some animals, and destruction of plant cell walls. Gene colocalization into a bicistronic operon observed for some PLPs and their inhibitors (as in the case of M4in) implies a mutually consistent functioning of both entities. The originality of the amino acid sequence of M4in suggests it belongs to a previously unknown protein family and this encourages structural studies. In this work, we report a near-complete assignment of

Here we report bisphenol derivatives of fluorene (BDFs) as a new type of chemical probes targeting a histone-like HU protein, a global regulator of bacterial nucleoids, via its dimerization interface perturbation. BDFs were identified by virtual screening and molecular docking that targeted the core of DNA-binding β-saddle-like domain of the HU protein from Spiroplasma melliferum. However, NMR spectroscopy, complemented with molecular dynamics and site-directed mutagenesis, indicated that the actual site of the inhibitors’ intervention consists of residues from the α-helical domain of one monomer and the side portion of the DNA-binding domain of another monomer. BDFs inhibited DNA-binding properties of HU proteins from mycoplasmas S. melliferum, Mycoplasma gallicepticum and Escherichia coli with half-maximum inhibitory concentrations in the range between 5 and 10 µM. In addition, BDFs demonstrated antimicrobial activity against mycoplasma species, but not against E. coli, which is consistent with the compensatory role of other nucleoid-associated proteins in the higher bacteria. Further evaluation of antimicrobial effects of BDFs against various bacteria and viruses will reveal their pharmacological potential, and the allosteric inhibition mode reported here, which avoids direct competition for the binding site with DNA, should be considered in the development of small molecule inhibitors of nucleoid-associated proteins as well as other types of DNA-binding multimeric proteins.

In aqueous medium, [Pr(H2O)(DOTA)]− (I) undergo a intramolecular interconversion between two conformers (here DOTA = 1, 4, 7, 10-tetraaza-1, 4, 7, 10-tetrakis (carboxymethyl)cyclododecan; n = 0, 1, 2). Two types of molecular dynamic processes were found {enantiomerization and interconversion of the square-anti-prismatic (SAP) to twisted-square-anti-prismatic conformers (TSAP)}. The free energies of activation of the SAP to TSAP interconversion (for I complex) are found to be 50 ± 3 and 54 ± 4 kJ mol−1 (according to 1H and 13C NMR respectively). And, the activation free energy of enantiomerization is ΔG≠298 = 65 ± 2 kJ mol−1. It was evaluated that the activation energy of enantiomerization in [Gd(H2O) (DOTA)]− (II) is 64 ± 2 kJ mol−1 (by analysis of the dependence of the free activation energy on the ionic radius of other lanthanide ions). The experimental paramagnetic LIS values of I are well fitted by linear dependence on 1/T. It was found that I may be used as NMR thermometric sensors for controlling the local temperature in aqueous medium.

The reaction of spillover hydrogen (SH) with cyclopropylglycine (cPG) in high-temperature solid-state catalytic hydrogen isotope exchange (HTCIE) has been studied experimentally and using density functional theory (DFT). NMR spectroscopy and mass spectrometry have shown a high regioselectivity and stereoselectivity for the reaction of spillover hydrogen with cPG fragments. The Gly fragment has been shown to be the most reactive in HTCIE, for which high stereoselectivity of substitution of hydrogen by deuterium has been demonstrated.

Single-point mutations in the transmembrane (TM) region of receptor tyrosine kinases (RTKs) can lead to abnormal ligand-independent activation. We use a combination of computational modeling, NMR spectroscopy and cell experiments to analyze in detail the mechanism of how TM domains contribute to the activation of wild-type (WT) PDGFRA and its oncogenic V536E mutant. Using a computational framework, we scan all positions in PDGFRA TM helix for identification of potential functional mutations for the WT and the mutant and reveal the relationship between the receptor activity and TM dimerization via different interfaces. This strategy also allows us design a novel activating mutation in the WT (I537D) and a compensatory mutation in the V536E background eliminating its constitutive activity (S541G). We show both computationally and experimentally that single-point mutations in the TM region reshape the TM dimer ensemble and delineate the structural and dynamic determinants of spontaneous activation of PDGFRA via its TM domain. Our atomistic picture of the coupling between TM dimerization and PDGFRA activation corroborates the data obtained for other RTKs and provides a foundation for developing novel modulators of the pathological activity of PDGFRA.

Background Prior studies of the human growth hormone receptor (GHR) revealed a distinct role of spatial rearrangements of its dimeric transmembrane domain in signal transduction across membrane. Detailed structural information obtained in the present study allowed elucidating the bases of such rearrangement and provided novel insights into receptor functioning. Methods We investigated the dimerization of recombinant TMD fragment GHR254–294 by means of high-resolution NMR in DPC micelles and molecular dynamics in explicit POPC membrane. Results We resolved two distinct dimeric structures of GHR TMD coexisting in membrane-mimicking micellar environment and providing left- and right-handed helix-helix association via different dimerization motifs. Based on the available mutagenesis data, the conformations correspond to the dormant and active receptor states and are distinguished by cis-trans isomerization of Phe-Pro266 bond in the transmembrane helix entry. Molecular dynamic relaxations of the structures in lipid bilayer revealed the role of the proline residue in functionally significant rearrangements of the adjacent juxtamembrane region supporting alternation between protein-protein and protein-lipid interactions of this region that can be triggered by ligand binding. Also, the importance of juxtamembrane S S bonding for signal persistency, and somewhat unusual aspects of transmembrane region interaction with water molecules were demonstrated. Conclusions Two alternative dimeric structures of GHR TMD attributed to dormant and active receptor states interchange via allosteric rearrangements of transmembrane helices and extracellular juxtamembrane regions that support coordination between protein-protein and protein-lipid interactions. General significance This study provides a holistic vision of GHR signal transduction across the membrane emphasizing the role of protein-lipid interactions.

In the case of soluble proteins, chemical shift mapping is used to identify the intermolecular interfaces when the NOE-based calculations of spatial structure of the molecular assembly are impossible or impracticable. However, the reliability of the membrane protein interface mapping based on chemical shifts or other relevant parameters was never assessed. In the present work, we investigate the predictive power of various NMR parameters that can be used for mapping of helix-helix interfaces in dimeric TM domains. These parameters are studied on a dataset containing three structures of helical dimers obtained for two different proteins in various membrane mimetics. We conclude that the amide chemical shifts have very little predictive value, while the methyl chemical shifts could be used to predict interfaces, though with great care. We suggest an approach based on conversion of the carbon NMR relaxation parameters of methyl groups into parameters of motion, and one of such values, the characteristic time of methyl rotation, appears to be a reliable sensor of interhelix contacts in transmembrane domains. The carbon NMR relaxation parameters of methyl groups can be measured accurately and with high sensitivity and resolution, making the proposed parameter a useful tool for investigation of protein-protein interfaces even in large membrane proteins. An approach to build the models of transmembrane dimers based on perturbations of methyl parameters and TMDOCK software is suggested.

Receptor tyrosine kinases (RTKs) occupy a separate functional niche among membrane receptors, which is determined by the special features of mechanisms of the signal transduction through a cellular membrane. RTKs are involved in the regulation of development and homeostasis of all the tissues of a human organism, playing a central role in cell proliferation, differentiation, and adhesion. A necessary condition of the biochemical signal transduction through a plasmatic membrane is a ligand-dependent or a ligand-independent dimerization (and/or an oligomerization) of RTKs which is accompanied by conformational rearrangements of all the RTK domains, including the α-helical transmembrane segments. In this review, the main aspects of structure-function relationship for RTKs from various receptor subfamilies are briefly discussed. It is shown in the light of the recently obtained biophysical and biochemical data that functioning of RTK receptors is mediated not only by protein–protein interactions, but by the state of the lipid environment as one of the main components of a self-consistent signal transduction system as well. The new principles of intercellular signal transduction through a membrane replenish the molecular mechanisms of the RTK functioning that have been earlier proposed and explain a number of paradoxes which are observed upon activation of wild-type receptors and the receptors with pathogenic transmembrane mutations. Understanding of the complex mechanisms of the signaling processes can facilitate the successful search for new opportunities of influence on the RTK biological functions with potential therapeutic consequences.

Among membrane receptors, the single-span receptor protein kinases occupy a broad but specific functional niche determined by distinctive features of the underlying transmembrane signaling mechanisms that are briefly overviewed on the basis of some of the most representative examples, followed by a more detailed discussion of several hierarchical levels of organization and interactions involved. All these levels, including single-molecule interactions (e.g., dimerization, liganding, chemical modifications), local processes (e.g. lipid membrane perturbations, cytoskeletal interactions), and larger scale phenomena (e.g., effects of membrane surface shape or electrochemical potential gradients) appear to be closely integrated to achieve the observed diversity of the receptor functioning. Different species of receptor protein kinases meet their specific functional demands through different structural features defining their responses to stimulation, but certain common patterns exist. Signaling by receptor protein kinases is typically associated with the receptor dimerization and clustering, ligand-induced rearrangements of receptor domains through allosteric conformational transitions with involvement of lipids, release of the sequestered lipids, restriction of receptor diffusion, cytoskeleton and membrane shape remodeling. Understanding of complexity and continuity of the signaling processes can help identifying currently neglected opportunities for influencing the receptor signaling with potential therapeutic implications. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.

The epidermal growth factor receptor (EGFR) family is an important class of receptor tyrosine kinases, mediating a variety of cellular responses in normal biological processes and in pathological states of multicellular organisms. Different modes of dimerization of the human EGFR transmembrane domain (TMD) in different membrane mimetics recently prompted us to propose a novel signal transduction mechanism based on protein-lipid interaction. However, the experimental evidence for it was originally obtained with slightly different TMD fragments used in the two different mimetics, compromising the validity of the comparison. To eliminate ambiguity, we determined the nuclear magnetic resonance (NMR) structure of the bicelle-incorporated dimer of the EGFR TMD fragment identical to the one previously used in micelles. The NMR results augmented by molecular dynamics simulations confirm the mutual influence of the TMD and lipid environment, as is required for the proposed lipid-mediated activation mechanism. They also reveal the possible functional relevance of a subtle interplay between the concurrent processes in the lipid and protein during signal transduction.

Introduction Erb-b2 receptor tyrosine kinase (HER2) transmembrane domain (TMD) mutations (HER2 V659E , HER2 G660D ) have previously been identified in lung adenocarcinomas, but their frequency and clinical significance is unknown. Methods We prospectively analyzed 8551 consecutive lung adenocarcinomas using hybrid capture–based comprehensive genomic profiling (CGP) at the request of the individual treating physicians for the purpose of making therapy decisions. Results We identified 15 cases (0.18%) of HER2 TMD mutations (HER2 V659E/D , HER2 G660D ) through CGP of 8551 lung adenocarcinomas. HER2 TMD mutations were mutually exclusive from HER2 kinase domain mutations and other oncogenic drivers in lung adenocarcinoma. Only two cases with HER2 TMD mutations (13%) had concurrent Erb-b2 receptor tyrosine kinase 2 gene ( HER2 ) amplification. Structural analysis of HER2 TMD association revealed that mutations at positions V659 and G660 to the highly polar residues glutamic acid, aspartic acid, or arginine should stabilize homodimerization and heterodimerization of HER2 in the active conformation. Treatment with afatinib, a pan-HER inhibitor, resulted in durable clinical response in three of four patients with lung adenocarcinoma, with two harboring HER2 V659E and one with double HER2 V659E/G660R mutations. HER2 TMD mutations (V659 and G660) are found in other non-NSCLC malignancies, and analogous TMD mutations are also found in EGFR, HER3, and HER4. Conclusion HER2 TMD mutations represent rare but distinct targetable driver mutations in lung adenocarcinoma. CGP capable of detecting diverse HER2 alterations, including HER2 TMD mutations, should be broadly adopted to identify all patients who may benefit from HER2-targeted therapies.

Insulin receptor–related receptor (IRR) is a receptor tyrosine kinase of the insulin receptor family and functions as an extracellular alkali sensor that controls metabolic alkalosis in the regulation of the acid–base balance. In the present work, we sought to analyze structural features of IRR by comparing them with those of the insulin receptor, which is its closest homolog but does not respond to pH changes. Using small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM), we investigated the overall conformation of the recombinant soluble IRR ectodomain (ectoIRR) at neutral and alkaline pH. In contrast to the well-known inverted U-shaped (or λ-shaped) conformation of the insulin receptor, the structural models reconstructed at different pH values revealed that the ectoIRR organization has a "droplike" shape with a shorter distance between the fibronectin domains of the disulfide-linked dimer subunits within ectoIRR. We detected no large-scale pH-dependent conformational changes of ectoIRR in both SAXS and AFM experiments, an observation that agreed well with previous biochemical and functional analyses of IRR. Our findings indicate that ectoIRR's sensing of alkaline conditions involves additional molecular mechanisms, for example engagement of receptor juxtamembrane regions or the surrounding lipid environment.

Structural investigations need ready supply of the isotope labeled proteins with inserted mutations n the quantities sufficient for the heteronuclear NMR. Though cell-free expression system has been widely used in the past years, high startup cost and complex compound composition prevent many researches from the developing this technique, especially for membrane protein production. Here we demonstrate the utility of a robust, cost-optimized cell-free expression technique for production of the physiologically important transmembrane fragment of amyloid precursor protein, APP 686-726 , containing Alzheimer's disease mutations in the juxtamembrane (E693G, Arctic form) and the transmembrane parts (V717G, London form, or L723P, Australian form). The protein cost was optimized by varying the FM/RM ratio as well as the amino acid concentration. We obtained the wild-type and mutant transmembrane fragments in the pellet mode of continuous exchange cell-free system consuming only commercial algal mixture of the 13 C, 15 N-labeled amino acids. Scaling up analytical tests, we achieved milligram quantity yields of isotope labeled wild-type and mutant APP 686-726 for structural studies by high resolution NMR spectroscopy in membrane mimicking environment. The described approach has from 5 to 23-fold cost advantage over the bacterial expression methods described earlier and 1.5 times exceeds our previous result obtained with the longer APP 671-726 WT fragment.

The EGFR/HER receptor family of an epidermal growth factor represents an important class of the receptor tyrosine kinases playing the key role in the control of cell growth and differentiation in mammalian cells, as well as in the development of a number of pathological processes, including oncogenesis. Binding of a ligand to the extracellular domains initiates switching of the EGFR/HER receptor between the alternative dimeric states that causes the allosteric activation of kinase domains in cell cytoplasm. The transmembrane (TM) domain and adjacent flexible regions alternatively interacting with either membrane surface or kinase domains are directly involved in the complex conformational transition in EGFR/HERs. Here we report on a highly efficient system of the cell free production of the EGFR/HER TM domains with functionally important juxtamembrane (JM) regions for the investigation of the molecular basis of biochemical signal transduction across the cell membrane. To increase the efficiency of synthesis of the EGFR/HER TM-JM fragments of the receptors, we used two N-terminal expression tags, which significantly increased the protein yield. In the case of the TM-JM fragments of EGFR (residues 638–692) and HER2 (residues 644–700), the method allowed us to obtain milligram quantities of the 13C,15N-labeled protein for structural and biophysical investigations in the membrane-mimicking environments using high-resolution heteronuclear NMR spectroscopy.

The insulin receptor (IR), insulin-like growth factor 1 receptor (IGF-1R), and insulin receptor-related receptor (IRR) form a mini family of predimerized receptor-like tyrosine kinases. IR and IGF-1R bind to their peptide agonists triggering metabolic and cell growth responses. In contrast, IRR, despite sharing with them a strong sequence homology, has no peptide-like agonist but can be activated by mildly alkaline media. The spatial structure and activation mechanisms of IRR have not been established yet. The present work represents the first account of a structural analysis of a predimerized receptor-like tyrosine kinase by high-resolution atomic force microscopy in their basal and activated forms. Our data suggest that in neutral media, inactive IRR has two conformations, where one is symmetrical and highly similar to the inactive Λ/U-shape of IR and IGF-1R ectodomains, whereas the second is drop-like and asymmetrical resembling the IRR ectodomain in solution. We did not observe complexes of IRR intracellular catalytic domains of the inactive receptor forms. At pH 9.0, we detected two presumably active IRR conformations, Γ-shaped and T-shaped. Both of conformations demonstrated formation of the complex of their intracellular catalytic domains responsible for autophosphorylation. The existence of two active IRR forms correlates well with the previously described positive cooperativity of the IRR activation. In conclusion, our data provide structural insights into the molecular mechanisms of alkali-induced IRR activation under mild native conditions that could be valuable for interpretation of results of IR and IGF-IR structural studies.

Despite the biological significance of insulin signaling, the molecular mechanisms of activation of the insulin receptor (IR) and other proteins from its family remain elusive. Current hypothesis on signal transduction suggests ligand-triggered structural changes in the extracellular domain followed by transmembrane (TM) domains closure and dimerization leading to trans-autophosphorylation and kinase activity in intracellular segments of the receptor. Using NMR spectroscopy, we detected dimerization of isolated TM segments of IR in different membrane-mimicking environments and observed multiple signals of NH groups of protein backbone possibly corresponding to several dimer conformations. Taking available experimental data as constraints, several atomistic models of dimeric TM domains of IR and insulin-like growth factor 1 (IGF-1R) receptors were elaborated. Molecular dynamics simulations of IR ectodomain revealed noticeable collective movements potentially responsible for closure of the C-termini of FnIII-3 domains and spatial approaching of TM helices upon insulin-induced receptor activation. In addition, we demonstrated that the intracellular part of the receptor does not impose restrictions on the positioning of TM helices in the membrane. Finally, we used two independent structure prediction methods to generate a series of dimer conformations followed by their cluster analysis and dimerization free energy estimation to select the best dimer models. Biological relevance of the later was further tested via comparison of the hydrophobic organization of TM helices for both wild-type receptors and their mutants. Based on these data, the ability of several segments from other proteins to functionally replace IR and/or IGF-1R TM domains was explained.

Side chains possess a broader conformational space (compared to the backbone) and are directly affected by intra- and intermolecular interactions, hence their dynamics and the corresponding NMR relaxation data are more sensitive and informative. Nevertheless, transverse relaxation in [Formula: see text] ([Formula: see text] or [Formula: see text]) spin systems is predominantly non-measurable in uniformly [Formula: see text]-labeled proteins due to cross-correlation effects. In the present publication, we propose a number of pulse sequences for accurate and precise measurement of the dipole-dipole transverse cross-correlated relaxation rate [Formula: see text], which, similarly to [Formula: see text] measurements, provides information about the amplitudes of intramolecular dynamics. The suggested approach has allowed us to circumvent a number of obstacles that were limiting earlier applications of [Formula: see text]: (1) impossibility of transmission of the central component of the triplet of [Formula: see text] group to [Formula: see text]-acquisition via INEPT has been solved by transmission of the averaged signal of "inner" and "outer" components of the triplet; (2) direct recording of the entire triplets resulting in substantial overlap of side chain signals has been replaced by recording of individual singlets with the use of [Formula: see text]-modulated approach and constant-time evolution; (3) low sensitivity has been enhanced via proton acquisition which required special attention to a zero-quantum coherence evolution. The proposed method expands the set of "dynamics sensors" covering protein side chains and substantially improves the quality and the level of detail of experimental data describing dynamic processes in proteins and protein complexes.

Growth hormone somatotropin and its membrane receptor GHR, belonging to a superfamily of the type I receptors possessing tyrosine kinase activity, are involved in the intercellular signal transduction cascade and regulate a number of important physiological and pathological processes in humans. Binding with somatotropin triggers a transition of GHR between two alternative dimer states, resulting in an allosteric activation of JAK2 tyrosine kinase in the cell cytoplasm. Transmembrane domain of GHR directly involved in this complex conformational transition. It has presumably two dimerization interfaces corresponding to the "unliganded" and the active state of GHR. In order to study the molecular basis of biochemical signal transduction mechanism across the cell membrane, we have developed an efficient cell-free production system of a TM fragment of GHR, which contains its TM domain flanked by functionally important juxtamembrane regions (GHRtm residues 254-298). The developed system allows to obtain -1 mg per 1 ml of reaction mixture of 13C- and 15N-isotope-labeled protein for structural and dynamic studies of the GHR TM domain dimerization in the membrane-mimicking medium by high-resolution heteronuclear NMR spectroscopy.

The goal of the study was to elaborate the pharmacokinetics methods of the amide derivative of peptide HLDF-6 (TGENHR-NH2) and its range of nootropic and neuroprotective activity is wide. The hexapeptide 41TGENHR46 is a fragment of the HDLF differentiation factor. It forms the basis for the development of preventive and therapeutic preparations for treating cerebrovascular and neurodegenerative conditions. Pharmacokinetic and molecular mechanisms of the action of the HLDF-6 peptide were studied using tritium- and deuterium-labeled derivatives of this peptide, produced with the use of the high-temperature solid-state catalytic isotope exchange reaction (HSCIE). This reaction was employed to produce the tritium-labeled peptide [3H]TGENHR-NH2 with a molar radioactivity of 230 Ci/mmol and the deuterium-labeled peptide [2H]TGENHR-NH2 with an average deuterium incorporation equal to 10.5 atoms. It was shown by the NMR spectroscopy that the isotope label distribution over the labeled peptide's molecule was uniform, which allowed qualitative analysis ofboth the peptide itself and its fragments in the organism's tissues to be conducted. The newly developed pharmacokinetics method makes it possible to avoid almost completely losses of the peptides under study due to biodegradation during the analysis of tissues. These labeled peptides were used in mice, rats and rabbits to study the pharmacokinetics of the peptide and to calculate the values of its principal pharmacokinetic parameters. Characteristics of its pharmacokinetic profile in the blood were obtained, the hypothesis of pharmacokinetics linearity tested, its metabolism analyzed and its bioavailability value, 34%, calculated. It has been shown that the studied TGENHR-NH2 peptide shows high resistance to hydrolysis in the blood plasma, with dipeptidyl aminopeptidases making the largest contribution to its hydrolysis.

The histone-like (HU) protein is one of the major nucleoid-associated proteins of the bacterial nucleoid, which shares high sequence and structural similarity with IHF but differs from the latter in DNA-specificity. Here, we perform an analysis of structural-dynamic properties of HU protein from Spiroplasma melliferum and compare its behavior in solution to that of another mycoplasmal HU from Mycoplasma gallisepticum. The high-resolution heteronuclear NMR spectroscopy was coupled with molecular-dynamics study and comparative analysis of thermal denaturation of both mycoplasmal HU proteins. We suggest that stacking interactions in two aromatic clusters in the HUSpm dimeric interface determine not only high thermal stability of the protein, but also its structural plasticity experimentally observed as slow conformational exchange. One of these two centers of stacking interactions is highly conserved among the known HU and IHF proteins. Second aromatic core described recently in IHFs and IHF-like proteins is considered as a discriminating feature of IHFs. We performed an electromobility shift assay to confirm high affinities of HUSpm to both normal and distorted dsDNA, which are the characteristics of HU protein. MD simulations of HUSpm with alanine mutations of the residues forming the non-conserved aromatic cluster demonstrate its role in dimer stabilization, as both partial and complete distortion of the cluster enhances local flexibility of HUSpm.

Mature transforming growth factor beta1 (TGF-β1) is a homodimeric protein with a single disulfide bridge between Cys77 on the respective monomers. The synthetic DNA sequence encoding the mature human TGF-β1/C77S (further termed TGF-β1m) was cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin (Trx) immediately after the DNA sequence encoding enteropeptidase recognition site. High-level expression (~1.5 g l(-1)) of Trx/TGF-β1m fusion was achieved in Escherichia coli BL21(DE3) strain mainly in insoluble form. The fusion was solubilized and refolded in glutathione redox system in the presence of zwitterionic detergent CHAPS. After refolding, Trx/TGF-β1m fusion was cleaved by enteropeptidase, and the carrier protein of TGF-β1m was separated from thioredoxin on Ni-NTA agarose. Separation of monomeric molecules from the noncovalently bounded oligomers was done using cation-exchange chromatography. The structure of purified TGF-β1m was confirmed by circular dichroism analysis. The developed technology allowed purifying biologically active tag-free monomeric TGF-β1m from bacteria with a yield of about 2.8 mg from 100 ml cell culture. The low-cost and easy purification steps allow considering that our proposed preparation of recombinant monomeric TGF-β1 could be employed for in vitro and in vivo experiments as well as for therapeutic intervention.

Fibroblast growth factor receptor 3 (FGFR3) transduces biochemical signals via lateral dimerization in the plasma membrane, and plays an important role in human development and disease. At least 8 different pathogenic mutations, implicated in cancers and growth disorders, have been identified in FGFR3 transmembrane segment. Here we use heteronuclear NMR spectroscopy to determine the dimeric structure of FGFR3 transmembrane domain in membrane-mimicking DPC/SDS (9/1) micelles. In the structure, the two transmembrane helices pack into a symmetric left-handed dimer, with intermolecular stacking interactions occurring in the dimer central region. Some pathogenic mutations fall within the helix-helix interface, while others are located within a putative alternative interface. This implies that while the observed dimer structure is important for FGFR3 signaling, the mechanism of FGFR3-mediated transduction across the plasma membrane is complex. We propose a FGFR3 signaling mechanism that is based on the solved structure, available structures of isolated soluble FGFR domains, and published biochemical and biophysical data.

Solid-state catalytic isotope exchange of hydrogen under the action of spillover hydrogen in organic compounds applied on a nonorganic carrier has been studied. The deuterium-labeled peptide [D]dalargin containing 14 deuterium atoms, [D]melatonin, and [D]histamine with the 72–92% substitution degree of all C–H bonds were prepared by this reaction. The activation energy of hydrogen isotope exchange with T2 and D2 in glycine and α-aminoisobutyric acid was obtained experimentally. It was shown that for the studied reaction the kinetic isotopic effect is 1.2–1.4 by the Hartree–Fock method, which is several times smaller than one in the liquid-phase reactions. Quantum chemical calculations of the isotope shift values in the electronic spectra of deuterium-labeled metal ion complexes were performed using the RB3LYP/LanL2DZ and CIS/LanL2DZ methods for calculation of the ground and the excited states. It was shown for deuterium-labeled histamine complexes [D12]Pd(him)2Cl2 and [D12]Cu(him)2Cl that the isotopic eff...

Mutations in transmembrane (TM) domains of receptor tyrosine kinases are shown to cause a number of inherited diseases and cancer development. Here, we use a combined molecular modeling approach to understand molecular mechanism of effect of G380R and A391E mutations on dimerization of TM domains of human fibroblast growth factor receptor 3 (FGFR3). According to results of Monte Carlo conformational search in the implicit membrane and further molecular dynamics simulations, TM dimer of this receptor is able to form a number of various conformations, which differ significantly by the free energy of association in a full-atom model bilayer. The aforementioned mutations affect dimerization efficiency of TM segments and lead to repopulation of conformational ensemble for the dimer. Particularly, both mutations do not change the dimerization free energy of the predominant (putative "non-active") symmetric conformation of TM dimer, while affect dimerization efficiency of its asymmetric ("intermediate") and alternative symmetric (putative "active") models. Results of our simulations provide novel atomistic prospective of the role of G380 and A391E mutations in dimerization of TM domains of FGFR3 and their consecutive contributions to the activation pathway of the receptor.

Receptor tyrosine kinases (RTKs) play an important role in intercellular signal transduction through the plasma membrane. RTKs are integral membrane proteins activated upon lateral homo- or heterodimerization involving their transmembrane domain. The polymorphism and mutations in RTK transmembrane (TM) domains are directly associated with a number of human diseases. The family of epidermal growth factor receptors, ErbB, is an important class of RTKs participating in human cell growth, development, and differentiation. In order to investigate the influence of pathogenic mutations in ErbB TM domains on the structural and dynamic properties of these receptors and on specific interactions of their TM domains, we have developed highly effective systems of bacterial expression and purification of recombinant transmembrane fragments ErbB2641–684 with pro-oncogenic substitution of Val659 by Glu or Gln. Transmembrane fragments were obtained in Escherichia coli BL21 (DE3) pLysS as a fusion protein with thioredoxin A. The purification protocol includes immobilized metal ion affinity chromatography (IMAC) and cation-exchange chromatography. The application of the protease Thrombin for hybrid protein hydrolysis considerably reduces financial expenditure as compared to the analogous protocols. The described techniques allow obtaining the milligram quantities of ErbB2 transmembrane fragments and its 15N-/[15N, 13C]-isotope-labeled derivatives for the analysis of their spatial structure using high-resolution heteronuclear NMR spectroscopy in a membrane-mimicking milieu.

Neuraminidase 1 (NEU1) is a lysosomal sialidase catalyzing the removal of terminal sialic acids from sialyloconjugates. A plasma membrane-bound NEU1 modulating a plethora of receptors by desialylation, has been consistently documented from the last ten years. Despite a growing interest of the scientific community to NEU1, its membrane organization is not understood and current structural and biochemical data cannot account for such membrane localization. By combining molecular biology and biochemical analyses with structural biophysics and computational approaches, we identified here two regions in human NEU1 - segments 139–159 (TM1) and 316–333 (TM2) - as potential transmembrane (TM) domains. In membrane mimicking environments, the corresponding peptides form stable α-helices and TM2 is suited for self-association. This was confirmed with full-size NEU1 by co-immunoprecipitations from membrane preparations and split-ubiquitin yeast two hybrids. The TM2 region was shown to be critical for dimerization since introduction of point mutations within TM2 leads to disruption of NEU1 dimerization and decrease of sialidase activity in membrane. In conclusion, these results bring new insights in the molecular organization of membrane-bound NEU1 and demonstrate, for the first time, the presence of two potential TM domains that may anchor NEU1 in the membrane, control its dimerization and sialidase activity.

The histone-like (HU) protein is one of the major nucleoid-associated proteins involved in DNA supercoiling and compaction into bacterial nucleoid as well as in all DNA-dependent transactions. This small positively charged dimeric protein binds DNA in a non-sequence specific manner promoting DNA super-structures. The majority of HU proteins are highly conserved among bacteria; however, HU protein from Mycoplasma gallisepticum (HUMgal) has multiple amino acid substitutions in the most conserved regions, which are believed to contribute to its specificity to DNA targets unusual for canonical HU proteins. In this work, we studied the structural dynamic properties of the HUMgal dimer by NMR spectroscopy and MD simulations. The obtained all-atom model displays compliance with the NMR data and confirms the heterogeneous backbone flexibility of HUMgal. We found that HUMgal, being folded into a dimeric conformation typical for HU proteins, has a labile α-helical body with protruded β-stranded arms forming DNA-binding domain that are highly flexible in the absence of DNA. The amino acid substitutions in conserved regions of the protein are likely to affect the conformational lability of the HUMgal dimer that can be responsible for complex functional behavior of HUMgal in vivo, e.g. facilitating its spatial adaptation to non-canonical DNA-targets.

ErbB is a family of epidermal growth factor receptors representing an important class of receptor tyrosine kinases that play a leading role in cellular growth, development, and differentiation. Transmembrane domains of these receptors transduce biochemical signals across the plasma membrane via lateral homo- and heterodimerization. The relatively small size of ErbB transmembrane domain complexes with detergents or lipids makes it possible to study their detailed spatial structure using three-dimensional heteronuclear high-resolution NMR spectroscopy. Here, we describe an efficient expression system and a purification procedure for preparative-scale production of transmembrane peptides from all four ErbB proteins—ErbB1, ErbB2, ErbB3, and ErbB4—for the purpose of structural studies. The recombinant peptides were produced in Escherichia coli BL21(DE3)pLysS cells as N-terminal extensions of thioredoxin A. The fusion proteins were cleaved with the light chain of human enterokinase. Several (10–30) milligrams of purified isotope-labeled transmembrane peptides were isolated using a simple and convenient procedure, which consists of consecutive steps of immobilized metal affinity chromatography and cation-exchange chromatography. The purified peptides were reconstituted in a lipid/detergent environment (micelles or bicelles) and characterized using dynamic light scattering and CD and NMR spectroscopy. The data obtained indicate that purified ErbB transmembrane peptides are suitable for structural and dynamic studies of their homo- and heterodimer complexes using high resolution NMR spectroscopy.

The fibroblast growth factor receptor 3 (FGFR3) is a protein belonging to the family of receptor tyro- sine kinases. FGFR3 plays an important role in human skeletal development. Mutations in this protein, including Gly380Arg or Ala391Glu substitutions in the transmembrane (TM) region, can cause different disorders in bone development. The determination of the spatial structure of the FGFR3 TM domain in a normal protein and in a protein with single Gly380Arg and Ala391Glu mutations is essential in order to understand the mechanisms that control dimerization and signal transduction by receptor tyrosine kinases. The effective system of expression of eukaryotic genes in bacteria and the purification protocol for the production of milligram amounts of both normal TM fragments of FGFR3 and those with single pathogenic mutations Gly380Arg and Ala391Glu, as well as their 15 N- and ( 15 N, 13 C)-isotope-labelled derivatives, were described. Each peptide was produced in Escherichia coli BL21(DE3)pLysS cells as a C-terminal extension of thioredoxin A. The purification protocol involved im- mobilized metal affinity chromatography and cation- and anion-exchange chromatography, as well as the fusion protein cleavage with the light subunit of human enterokinase. The efficiency of the incorporation of target peptides into DPC/SDS and DPC/DPG micelles was confirmed using NMR spectroscopy. The described meth- odology of production of the native FGFR3 TM domain in norma and with single Gly380Arg and Ala391Glu mutations enables one to study their spatial structure using high-resolution heteronuclear NMR spectroscopy. KEYWORDS membrane protein; FGFR; bacterial expression; purification; detergent solubilization; NMR. ABBREVIATIONS FGFR - Fibroblast growth factor receptor family; FGFR3 - Fibroblast growth factor receptor 3; RTK - receptor tyrosine kinase; TM - transmembrane (domain of membrane protein); DPC — dodecylphospho - choline; DPG — dodecylphosphoglycerol; SDS - sodium dodecyl sulfate; TFE — 2,2,2-trifluoroethanol.