Nuclear Magnetic Resonance Solution Structure Research Articles

High-Resolution NMR Structures of Intrastrand Hairpins Formed by CTG Trinucleotide Repeats.

The CAG and CTG trinucleotide repeat expansions cause more than 10 human neurodegenerative diseases. Intrastrand hairpins formed by trinucleotide repeats contribute to repeat expansions, establishing them as potential drug targets. High-resolution structural determination of CAG and CTG hairpins poses as a long-standing goal to aid drug development, yet it has not been realized due to the intrinsic conformational flexibility of repetitive sequences. We herein investigate the solution structures of CTG hairpins using nuclear magnetic resonance (NMR) spectroscopy and found that four CTG repeats with a clamping G-C base pair was able to form a stable hairpin structure. We determine the first solution NMR structure of dG(CTG)4C hairpin and decipher a type I folding geometry of the TGCT tetraloop, wherein the two thymine residues form a T·T loop-closing base pair and the first three loop residues continuously stack. We further reveal that the CTG hairpin can be bound and stabilized by a small-molecule ligand, and the binding interferes with replication of a DNA template containing CTG repeats. Our determined high-resolution structures lay an important foundation for studying molecular interactions between native CTG hairpins and ligands, and benefit drug development for trinucleotide repeat expansion diseases.

Hard nut to crack: Solving the disulfide linkage pattern of the Neosartorya (Aspergillus) fischeri antifungal protein 2.

As a consequence of the fast resistance spreading, a limited number of drugs are available to treat fungal infections. Therefore, there is an urgent need to develop new antifungal treatment strategies. The features of a disulfide bond-stabilized antifungal protein, NFAP2 secreted by the mold Neosartorya (Aspergillus) fischeri render it to be a promising template for future protein-based antifungal drug design, which requires knowledge about the native disulfide linkage pattern as it is one of the prerequisites for biological activity. However; in the lack of tryptic and chymotryptic proteolytic sites in the ACNCPNNCK sequence, the determination of the disulfide linkage pattern of NFAP2 is not easy with traditional mass spectrometry-based methods. According to in silico predictions working with a preliminary nuclear magnetic resonance (NMR) solution structure, two disulfide isomers of NFAP2 (abbacc and abbcac) were possible. Both were chemically synthesized; and comparative reversed-phase high-performance liquid chromatography, electronic circular dichroism and NMR spectroscopy analyses, and antifungal susceptibility and efficacy tests indicated that the abbcac is the native pattern. This knowledge allowed rational modification of NAFP2 to improve the antifungal efficacy and spectrum through the modulation of the evolutionarily conserved γ-core region, which is responsible for the activity of several antimicrobial peptides. Disruption of the steric structure of NFAP2 upon γ-core modification led to the conclusions that this motif may affect the formation of the biologically active three-dimensional structure, and that the γ-core modulation is not an efficient tool to improve the antifungal efficacy or to change the antifungal spectrum of NFAP2. This article is protected by copyright. All rights reserved.

Solution NMR structure of cementum protein 1 derived peptide (CEMP1-p1) and its role in the mineralization process.

We report the characterization of the three-dimensional structure of the CEMP1-p1 peptide [MGTSSTDSQQAQHRRCSTSN: corresponding to residues 1-20 of the N-terminus of cementum protein 1 (CEMP1)]. This peptide imitates the capacity of CEMP1 to stimulate hydroxyapatite (HA) crystal nucleation and growth, and promotes the differentiation of periodontal ligament cells into a cementoblastic phenotype. Additionally, in experimental models of critical-sized calvarial defects in Wistar rats, CEMP1-p1 has shown osteogenic properties that enhanced the physiological deposition and maturation of newly formed bone. In this work, studies of CEMP1-p1 by circular dichroism (CD) and nuclear magnetic resonance (NMR) were performed in trifluoroethanol D2 (TFED2) and aqueous solution to determine the 3D structure of the peptide. Using the 3D model, experimental data from HA crystals formation and calcium fluorescence emission, we explain the biological mechanisms involved in CEMP1-p1 activity to promote calcium recruitment and its affinity to HA crystals. This information is valuable because it proposes, for the first time, a plausible molecular mechanism during the mineralization process, from a specific cementum protein-derived peptide.

Solution Nuclear Magnetic Resonance Structures of ATTTT and ATTTC Pentanucleotide Repeats Associated with SCA37 and FAMEs.

Expansions of ATTTT and ATTTC pentanucleotide repeats in the human genome are recently found to be associated with at least seven neurodegenerative diseases, including spinocerebellar ataxia type 37 (SCA37) and familial adult myoclonic epilepsy (FAME) types 1, 2, 3, 4, 6, and 7. The formation of non-B DNA structures during some biological processes is thought as a causative factor for repeat expansions. Yet, the structural basis for these pyrimidine-rich ATTTT and ATTTC repeat expansions remains elusive. In this study, we investigated the solution structures of ATTTT and ATTTC repeats using nuclear magnetic resonance spectroscopy. Here, we reveal that ATTTT and ATTTC repeats can form a highly compact minidumbbell structure at the 5'-end using their first two repeats. The high-resolution structure of two ATTTT repeats was determined, showing a regular TTTTA pentaloop and a quasi TTTT/A pentaloop. Furthermore, the minidumbbell structure could escape from proofreading by the Klenow fragment of DNA polymerase I when it was located at five or more base pairs away from the priming site, leading to a small-scale repeat expansion. Results of this work improve our understanding of ATTTT and ATTTC repeat expansions in SCA37 and FAMEs, and provide high-resolution structural information for rational drug design.

Ecdysis triggering hormone peptide in the African malaria mosquito Anopheles gambiae: The peptide structure for receptor activation.

Infections by mosquito-borne diseases represent one of the leading causes of death in third world countries. The rapid progression of resistance to conventional insecticide causes a significant threat to the highly efficient preventive methods currently in place. Insect neuropeptidergic system offers potential targets to control the insect vectors. The essential roles of the neuropeptide ecdysis triggering hormone (ETH) in insect development and reproduction led us to attempt understanding of the fundamentals of the biochemical interaction between ETH and its receptor in the African malaria mosquito Anopheles gambiae. One of two ETH peptides of the African malaria mosquito (AgETH1), a small peptide hormone with 17 amino acid residues (SESPGFFIKLSKSVPRI-NH2 ), was studied to elucidate its molecular structure. N-termini deletions and mutations of conserved amino acids in the ligand revealed the critical residues for the receptor activation. The solution structure of AgETH1 using 2D 1 H-1 H nuclear magnetic resonance (NMR) spectroscopy and nuclear overhauser effect (NOE) derived constraints revealed a short alpha helix between residues 3S and 11S. The NMR solution structure of AgETH1 will be of significant assistance for designing a new class of insecticidal compounds that acts on the AgETH receptor aiming for in silico docking studies.

NMR solution structures of d(GGCCTG)n repeats associated with spinocerebellar ataxia type 36

Expansion of d(GGCCTG)n hexanucleotide repeats in the NOP56 gene is the genetic cause of spinocerebellar ataxia type 36 (SCA36) which is an inheritable neurodegenerative disease. Non-B DNA is known to be the structural intermediate causing repeat expansions. Yet, the structure and mechanism of genetic instability of d(GGCCTG)n repeats remain elusive. In this work, we investigated the solution structures of sequences containing two to eight GGCCTG repeats using nuclear magnetic resonance (NMR) spectroscopy. They were found to form diverse secondary structures, including hairpin, duplex and G-quadruplex (G4). Intriguingly, the hairpin structure was present in all the investigated sequences. The NMR solution structure of the hairpin formed by d(GGCCTG)2 was determined, disclosing an unprecedented CCTGGG hexanucleotide loop in which the first and sixth loop residues formed a Watson-Crick loop-closing base pair, the second and third loop residues stacked in the major groove, whereas the fourth and fifth loop residues formed a G·G mismatch. Apart from the hairpin, antiparallel G4 and palindromic duplex structures were found to form in d(GGCCTG)2 and d(GGCCTG)3–8, respectively. Results of this work provide new insights into the genetic instability of d(GGCCTG)n repeats and structure-based drug design for SCA36.

Dynamic Na+/H+ exchanger 1 (NHE1) - calmodulin complexes of varying stoichiometry and structure regulate Ca2+-dependent NHE1 activation.

Calmodulin (CaM) engages in Ca2+-dependent interactions with numerous proteins, including a still incompletely understood physical and functional interaction with the human Na+/H+-exchanger NHE1. Using nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimetry, and fibroblasts stably expressing wildtype and mutant NHE1, we discovered multiple accessible states of this functionally important complex existing in different NHE1:CaM stoichiometries and structures. We determined the NMR solution structure of a ternary complex in which CaM links two NHE1 cytosolic tails. In vitro, stoichiometries and affinities could be tuned by variations in NHE1:CaM ratio and calcium ([Ca2+]) and by phosphorylation of S648 in the first CaM-binding α-helix. In cells, Ca2+-CaM-induced NHE1 activity was reduced by mimicking S648 phosphorylation and by mutation of the first CaM-binding α-helix, whereas it was unaffected by inhibition of Akt, one of several kinases phosphorylating S648. Our results demonstrate a diversity of NHE1:CaM interaction modes and suggest that CaM may contribute to NHE1 dimerization and thereby augment NHE1 regulation. We propose that a similar structural diversity is of relevance to many other CaM complexes.

NMR and MD simulations reveal the impact of the V23D mutation on the function of yeast oligosaccharyltransferase subunit Ost4.

Asparagine-linked glycosylation, also known as N-linked glycosylation, is an essential and highly conserved co- and post-translational protein modification in eukaryotes and some prokaryotes. In the central step of this reaction, a carbohydrate moiety is transferred from a lipid-linked donor to the side-chain of a consensus asparagine in a nascent protein as it is synthesized at the ribosome. Complete loss of oligosaccharyltransferase (OST) function is lethal in eukaryotes. This reaction is carried out by a membrane-associated multisubunit enzyme, OST, localized in the endoplasmic reticulum. The smallest subunit, Ost4, contains a single membrane-spanning helix that is critical for maintaining the stability and activity of OST. Mutation of any residue from Met18 to Ile24 of Ost4 destabilizes the enzyme complex, affecting its activity. Here, we report solution nuclear magnetic resonance structures and molecular dynamics (MD) simulations of Ost4 and Ost4V23D in micelles. Our studies revealed that while the point mutation did not impact the structure of the protein, it affected its position and solvent exposure in the membrane mimetic environment. Furthermore, our MD simulations of the membrane-bound OST complex containing either WT or V23D mutant demonstrated disruption of most hydrophobic helix-helix interactions between Ost4V23D and transmembrane TM12 and TM13 of Stt3. This disengagement of Ost4V23D from the OST complex led to solvent exposure of the D23 residue in the hydrophobic pocket created by these interactions. Our study not only solves the structures of yeast Ost4 subunit and its mutant but also provides a basis for the destabilization of the OST complex and reduced OST activity.

High-Resolution Structures of DNA Minidumbbells Comprising Type II Tetraloops with a Purine Minor Groove Residue.

Minidumbbell (MDB) is a newly discovered DNA structure formed by native sequences, which serves as a possible structural intermediate causing repeat expansion mutations in the genome and also a functional structural motif in constructing DNA-based molecular switches. Until now, all the reported MDBs containing two adjacent type II tetraloops were formed by pyrimidine-rich sequences 5'-YYYR YYYR-3' (Y and R represent pyrimidine and purine, respectively), wherein the second and sixth residues folded into the minor groove and interacted with each other. In this study, we have conducted a high-resolution nuclear magnetic resonance (NMR) spectroscopic investigation on alternative MDB-forming sequences and discovered that an MDB could also be formed stably with a purine in the minor groove, which has never been observed in any previously reported DNA type II tetraloops. Our refined NMR solution structures of the two MDBs formed by 5'-CTTG CATG-3' and 5'-CTTG CGTG-3' reveal that the sixth purine residue was driven into the minor groove via base-base stacking with the second thymine residue and adenine stacked better than guanine. The results of our present research work expand the sequence criteria for the formation of MDBs and shed light to explore the significance of MDBs.

Structural Characterization of the S-glycosylated Bacteriocin ASM1 from Lactobacillus plantarum

In order to protect their environmental niche, most bacteria secret antimicrobial substances designed to target specific bacterial strains that are often closely related to the producer strain. Bacteriocins, small, ribosomally synthesised antimicrobial peptides, comprise a class of such substances and can either inhibit (bacteriostatic) or kill (bactericidal) target cells. Glycocins are a class of bacteriocin that are post-translationally modified by one or more carbohydrate moieties that are either β-O-linked to either a serine or threonine and/or β-S-linked to a cysteine. The solution nuclear magnetic resonance structure (NMR) of the glycocin ASM1 (produced by Lactobacillus plantarum A-1), an orthologue of GccF, has been determined. In both structures, the disulfide bonds are essential for activity and restrict the mobility of the N-acetyl-glucosamine (GlcNAc) attached to Ser-18 (O-linked), compared to the much more flexible GlcNAc moiety on Cys-43 (S-linked). Interestingly, despite 88% sequence identity, the helical structure of ASM1 is less pronounced which appears to be consistent with the far ultra-violet circular dichroism (UV CD) spectra.

The Solution Structures and Interaction of SinR and SinI: Elucidating the Mechanism of Action of the Master Regulator Switch for Biofilm Formation in Bacillus subtilis

Bacteria have developed numerous protection strategies to ensure survival in harsh environments, with perhaps the most robust method being the formation of a protective biofilm. In biofilms, bacterial cells are embedded within a matrix that is composed of a complex mixture of polysaccharides, proteins, and DNA. The gram-positive bacterium Bacillus subtilis has become a model organism for studying regulatory networks directing biofilm formation. The phenotypic transition from a planktonic to biofilm state is regulated by the activity of the transcriptional repressor, SinR, and its inactivation by its primary antagonist, SinI. In this work, we present the first full-length structural model of tetrameric SinR using a hybrid approach combining high-resolution solution nuclear magnetic resonance (NMR), chemical cross-linking, mass spectrometry, and molecular docking. We also present the solution NMR structure of the antagonist SinI dimer and probe the mechanism behind the SinR-SinI interaction using a combination of biochemical and biophysical techniques. As a result of these findings, we propose that SinI utilizes a residue replacement mechanism to block SinR multimerization, resulting in diminished DNA binding and concomitant decreased repressor activity. Finally, we provide an evidence-based mechanism that confirms how disruption of the SinR tetramer by SinI regulates gene expression.

Nuclear magnetic resonance solution structure of Pisum sativum defensin 2 provides evidence for the presence of hydrophobic surface-clusters.

Pisum sativum defensin 2 (Psd2) is a small (4.7 kDa) antifungal peptide whose structure is held together by four conserved disulfide bridges. Psd2 shares the cysteine-stabilized alpha-beta (CSαβ) fold, which lacks a regular hydrophobic core. All hydrophobic residues are exposed to the surface, except for leucine 6. They are clustered in the surface formed by two loops, between β1 and α-helix and β2 and β3 sheets. The observation of surface hydrophobic clusters reveals a remarkable evolution of the CSαβ fold to expose and reorganize hydrophobic residues, which facilitates creating versatile binding sites.

Nuclear Magnetic Resonance Solution Structure and Functional Behavior of the Human Proton Channel.

The human voltage-gated proton channel [Hv1(1) or VSDO(2)] plays an important role in the human innate immune system. Its structure differs considerably from those of other cation channels. It is built solely of a voltage-sensing domain and thus lacks the central pore domain, which is essential for other cation channels. Here, we determined the solution structure of an N- and C-terminally truncated human Hv1 (Δ-Hv1) in the resting state by nuclear magnetic resonance (NMR) spectroscopy. Δ-Hv1 comprises the typical voltage-sensing antiparallel four-helix bundle (S1-S4) preceded by an amphipathic helix (S0). The solution structure corresponds to an intermediate state between resting and activated forms of voltage-sensing domains. Furthermore, Zn2+-induced closing of proton channel Δ-Hv1 was studied with two-dimensional NMR spectroscopy, which showed that characteristic large scale dynamics of open Δ-Hv1 are absent in the closed state of the channel. Additionally, pH titration studies demonstrated that a higher H+ concentration is required for the protonation of side chains in the Zn2+-induced closed state than in the open state. These observations demonstrate both structural and dynamical changes involved in the process of voltage gating of the Hv1 channel and, in the future, may help to explain the unique properties of unidirectional conductance and the exceptional ion selectivity of the channel.

Helical Structure of Recombinant Melittin.

Melittin is an extensively studied, 26-residue toxic peptide from honey bee venom. Because of its versatility in adopting a variety of secondary (helix or coil) and quaternary (monomer or tetramer) structures in various environments, melittin has been the focus of numerous investigations as a model peptide in protein folding studies as well as in studies involving binding to proteins, lipids, and polysaccharides. A significant body of evidence supports the view that melittin binds to these macromolecules in a predominantly helical conformation, but detailed structural knowledge of this conformation is lacking. In this report, we present nuclear magnetic resonance (NMR)-based structural insights into the helix formation of recombinant melittin in the presence of trifluoroethanol (TFE): a known secondary structure inducer in peptides. These studies were performed at neutral pH, with micromolar amounts of the peptide. Using nuclear Overhauser effect (NOE)-derived distance restraints from three-dimensional NMR spectra, we determined the atomic resolution solution NMR structure of recombinant melittin bearing a TFE-stabilized helix. To circumvent the complications with structure determination of small peptides with high conformational flexibility, we developed a workflow for enhancing proton NOEs by increasing the viscosity of the medium. In the TFE-containing medium, recombinant monomeric melittin forms a long, continuous helical structure, which consists of the N- and C-terminal α-helices and the noncanonical 310-helix in the middle. The noncanonical 310-helix is missing in the previously solved X-ray structure of tetrameric melittin and the NMR structure of melittin in methanol. Melittin's structure in TFE-containing medium provides insights into melittin's conformational transitions, which are relevant to the peptide's interactions with its biological targets.

NMR solution structure of an asymmetric intermolecular leaped V-shape G-quadruplex: selective recognition of the d(G2NG3NG4) sequence motif by a short linear G-rich DNA probe.

Aside from classical loops among G-quadruplexes, the unique leaped V-shape scaffold spans over three G-tetrads, without any intervening residues. This scaffold enables a sharp reversal of two adjacent strand directions and simultaneously participates in forming the G-tetrad core. These features make this scaffold itself distinctive and thus an essentially more accessible target. As an alternative to the conventional antisense method using a complementary chain, forming an intermolecular G-quadruplex from two different oligomers, in which the longer one as the target is captured by a short G-rich fragment, could be helpful for recognizing G-rich sequences and structural motifs. However, such an intermolecular leaped V-shape G-quadruplex consisting of DNA oligomers of quite different lengths has not been evaluated. Here, we present the first nuclear magnetic resonance (NMR) study of an asymmetric intermolecular leaped V-shape G-quadruplex assembled between an Oxytricha nova telomeric sequence d(G2T4G4T4G4) and a single G-tract fragment d(TG4A). Furthermore, we explored the selectivity of this short fragment as a potential probe, examined the kinetic discrimination for probing a specific mutant, and proposed the key sequence motif d(G2NG3NG4) essential for building the leaped V-shape G-quadruplexes.

Solution NMR structure of CHU_1110 from Cytophaga hutchinsonii, an AHSA1 protein potentially involved in metal ion stress response.

We report the solution nuclear magnetic resonance (NMR) structure of CHU_1110 from Cytophaga hutchinsonii. CHU_1110 contains three α-helices and one antiparallel β-sheet, forming a large cavity in the center of the protein, which are consistent with the structural characteristics of AHSA1 protein family. This protein shows high structural similarities to the prokaryotic proteins RHE_CH02687 from Rhizobium etli and YndB from Bacillus subtilis, which can bind with flavinoids. Unlike these two homologs, CHU_1110 shows no obvious interaction with flavonoids in NMR titration experiments. In addition, no direct interaction has been observed between CHU_1110 and ATP, although many homologous sequences of CHU_1110 have been annotated as ATPase. Combining the analysis of structural similarity of CHU_1110 and genomic context of its encoding gene, we speculate that CHU_1110 may be involved in the stress response of bacteria to heavy metal ions, even though its specific biological functions that need to be further investigated.

Structural Changes Induced by the Binding of the Calcium Desensitizer W7 to Cardiac Troponin.

Compounds that directly modulate the affinity of the thin filament calcium regulatory proteins in cardiac muscle have potential for treating heart disease. A recent "proof of concept" study showed that the desensitizer W7 can correct hyper-calcium-sensitive sarcomeres from RCM R193H inhibitory subunit troponin I (cTnI) transgenic mice. We have determined the high-resolution nuclear magnetic resonance solution structure of W7 bound to the regulatory domain of calcium binding subunit troponin C (cNTnC)-cTnI cChimera designed to represent the key aspects of the cTnC-cTnI interface. The structure shows that W7 does not perturb the overall structure of the cTnC-cTnI interface, with the helical structure and position of the cTnI switch region remaining intact upon W7 binding. The naphthalene ring of W7 sits in the hydrophobic pocket created by the cNTnC-cTnI switch peptide interface, while the positively charged amine tail extends into the solvent. The positively charged tail of W7 is in the proximity of Arg147 of the cTnI switch region, supporting the suggestion that electrostatic repulsion is an aspect underlying the mechanism of desensitization. Ser84 (replacing the unique Cys84 in cTnC reported to make a reversible covalent bond with levosimendan) also contacts W7.

Structural Basis for Rare Earth Element Recognition by Methylobacterium extorquens Lanmodulin.

Lanmodulin (LanM) is a high-affinity lanthanide (Ln)-binding protein recently identified in Methylobacterium extorquens, a bacterium that requires Lns for the function of at least two enzymes. LanM possesses four EF-hands, metal coordination motifs generally associated with CaII binding, but it undergoes a metal-dependent conformational change with a 100 million-fold selectivity for LnIIIs and YIII over CaII. Here we present the nuclear magnetic resonance solution structure of LanM complexed with YIII. This structure reveals that LanM features an unusual fusion of adjacent EF-hands, resulting in a compact fold to the best of our knowledge unique among EF-hand-containing proteins. It also supports the importance of an additional carboxylate ligand in contributing to the protein's picomolar affinity for LnIIIs, and it suggests a role of unusual N i+1-H···N i hydrogen bonds, in which LanM's unique EF-hand proline residues are engaged, in selective LnIII recognition. This work sets the stage for a detailed mechanistic understanding of LanM's Ln selectivity, which may inspire new strategies for binding, detecting, and sequestering these technologically important metals.

Structural Prediction of the Dimeric Form of the Mammalian Translocator Membrane Protein TSPO: A Key Target for Brain Diagnostics.

Positron emission tomography (PET) radioligands targeting the human translocator membrane protein (TSPO) are broadly used for the investigations of neuroinflammatory conditions associated with neurological disorders. Structural information on the mammalian protein homodimers—the suggested functional state of the protein—is limited to a solid-state nuclear magnetic resonance (NMR) study and to a model based on the previously-deposited solution NMR structure of the monomeric mouse protein. Computational studies performed here suggest that the NMR-solved structure in the presence of detergents is not prone to dimer formation and is furthermore unstable in its native membrane environment. We, therefore, propose a new model of the functionally-relevant dimeric form of the mouse protein, based on a prokaryotic homologue. The model, fully consistent with solid-state NMR data, is very different from the previous predictions. Hence, it provides, for the first time, structural insights into this pharmaceutically-important target which are fully consistent with experimental data.

The Crystal Structure of Gurmarin, a Sweet Taste-Suppressing Protein: Identification of the Amino Acid Residues Essential for Inhibition.

Gurmarin is a highly specific sweet taste-suppressing protein in rodents that is isolated from the Indian plant Gymnema sylvestre. Gurmarin consists of 35 amino acid residues containing 3 intramolecular disulfide bridges that form a cystine knot. Here, we report the crystal structure of gurmarin at a 1.45 Å resolution and compare it with previously reported nuclear magnetic resonance solution structures. The atomic structure at this resolution allowed us to identify a very flexible region consisting of hydrophobic residues. Some of these amino acid residues had been identified as a putative binding site for the rat sweet taste receptor in a previous study. By combining alanine-scanning mutagenesis of the gurmarin molecule and a functional cell-based receptor assay, we confirmed that some single point mutations in these positions drastically affect sweet taste receptor inhibition by gurmarin.