Crystal structure of the tandem phosphatase domains of RPTP LAR.

Most receptor-like protein tyrosine phosphatases (RPTPs) contain two conserved phosphatase domains (D1 and D2) in their intracellular region. The carboxy-terminal D2 domain has little or no catalytic activity. The crystal structure of the tandem D1 and D2 domains of the human RPTP LAR revealed that the tertiary structures of the LAR D1 and D2 domains are very similar to each other, with the exception of conformational differences at two amino acid positions in the D2 domain. Site-directed mutational changes at these positions (Leu-1644-to-Tyr and Glu-1779-to-Asp) conferred a robust PTPase activity to the D2 domain. The catalytic sites of both domains are accessible, in contrast to the dimeric blocked orientation model previously suggested. The relative orientation of the LAR D1 and D2 domains, constrained by a short linker, is stabilized by extensive interdomain interactions, suggesting that this orientation might be favored in solution.     

Photosystem II single crystals studied by transient EPR: the light-induced triplet state

Transient electron paramagnetic resonance (TR EPR) at 9.8 GHz has been used to study the light-induced triplet state in single crystals of Photosystem II (PS II). The crystals were grown from a solution of PS II core complexes from the thermophilic cyanobacterium Synechococcus elongatus. The core complexes contain at least 17 subunits, including the water-oxidizing complex, and 32 chlorophyll a molecules per PS II complex. The PS II complexes are active in light-induced electron transfer and water oxidation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with four dimers of PS II complexes per unit cell. Laser excitation was used to generate the recombination triplet state in PS II which was then studied by EPR at low temperatures (10 K). The crystal spectra show the same magnitude of the zero-field splitting (ZFS) values D, E as spectra obtained earlier for the triplet state of PS II in frozen solution. The orientation of the ZFS tensor D of the triplet state with respect to the crystallographic axes has been deduced from the analysis of angular-dependent EPR spectra. Knowledge of the orientation of the D tensor component perpendicular to the plane of the chlorophyll (D(Z)) allows an assignment on which chlorophyll of the reaction centre the triplet state is localized at low temperatures. Furthermore, the orientation of the D(X) and D(Y) components of the D tensor yielded the in-plane orientation of the respective chlorophyll in the reaction centre providing first experimental evidence for the orientation of this molecule in the PS II.     

Dilated Cardiomyopathy Mutations and Phosphorylation disrupt the Active Orientation of Cardiac Troponin C

Cardiac troponin (cTn) is made up of three subunits, cTnC, cTnI, and cTnT. The regulatory N-terminal domain of cTnC (cNTnC) controls cardiac muscle contraction in a calcium-dependent manner. We show that calcium-saturated cNTnC can adopt two different orientations, with the “active” orientation consistent with the 2020 cryo-EM structure of the activated cardiac thin filament by Yamada et al. Using solution NMR ¹⁵N R₂ relaxation analysis, we demonstrate that the two domains of cTnC tumble independently (average R₂ 10 s⁻¹), being connected by a flexible linker. However, upon addition of cTnI_1-77, the complex tumbles as a rigid unit (R₂ 30 s⁻¹). cTnI phosphomimetic mutants S22D/S23D, S41D/S43D and dilated cardiomyopathy- (DCM-)associated mutations cTnI K35Q, cTnC D75Y, and cTnC G159D destabilize the active orientation of cNTnC, with intermediate ¹⁵N R₂ rates (R₂ 17–23 s⁻¹). The active orientation of cNTnC is stabilized by the flexible tails of cTnI, cTnI_1-37 and cTnI_135-209. Surprisingly, when cTnC is incorporated into complexes lacking these tails (cTnC-cTnI_38-134, cTnC-cTnT_223-288, or cTnC-cTnI_38-134-cTnT_223-288), the cNTnC domain is still immobilized, revealing a new interaction between cNTnC and the IT-arm that stabilizes a “dormant” orientation. We propose that the calcium sensitivity of the cardiac troponin complex is regulated by an equilibrium between active and dormant orientations, which can be shifted through post-translational modifications or DCM-associated mutations.     

Induced fit in HIV-neutralizing antibody complexes: evidence for alternative conformations of the gp120 V3 loop and the molecular basis for broad neutralization

Human monoclonal antibody (mAb) 447-52D neutralizes a broad spectrum of HIV-1 isolates, whereas murine mAb 0.5beta, raised against gp120 of the X4 isolate HIV-1(IIIB), neutralizes this strain specifically. Two distinct gp120 V3 peptides, V3(MN) and V3(IIIB), adopt alternative beta-hairpin conformations when bound to 447-52D and 0.5beta, respectively, suggesting that the alternative conformations of this loop play a key role in determining the coreceptor specificity of HIV-1. To test this hypothesis and to better understand the molecular basis underlying an antibody's breadth of neutralization, the solution structure of the V3(IIIB) peptide bound to 447-52D was determined by NMR. V3(IIIB) and V3(MN) peptides bound to 447-52D exhibited the same N-terminal strand conformation, while the V3(IIIB) peptide revealed alternative N-terminal conformations when bound to 447-52D and 0.5beta. Comparison of the three known V3 structures leads to a model in which a 180 degrees change in the orientation of the side chains and the resulting one-residue shift in hydrogen bonding patterns in the N-terminal strand of the beta-hairpins markedly alter the topology of the surface that interacts with antibodies and that can potentially interact with the HIV-1 coreceptors. Predominant interactions of 447-52D with three conserved residues of the N-terminal side of the V3 loop, K312, I314, and I316, can account for its broad cross reactivity, whereas the predominant interactions of 0.5beta with variable residues underlie its strain specificity.     

NMR structure of the Vibrio vulnificus ribosomal protein S1 domains D3 and D4 provides insights into molecular recognition of single-stranded RNAs

The ribosomal S1 protein (rS1) is indispensable for translation initiation in Gram-negative bacteria. rS1 is a multidomain protein that acts as an RNA chaperone and ensures that mRNAs can bind the ribosome in a single-stranded conformation, which could be related to fast recognition. Although many ribosome structures were solved in recent years, a high-resolution structure of a two-domain mRNA-binding competent rS1 construct is not yet available. Here, we present the NMR solution structure of the minimal mRNA-binding fragment of Vibrio Vulnificus rS1 containing the domains D3 and D4. Both domains are homologues and adapt an oligonucleotide-binding fold (OB fold) motif. NMR titration experiments reveal that recognition of miscellaneous mRNAs occurs via a continuous interaction surface to one side of these structurally linked domains. Using a novel paramagnetic relaxation enhancement (PRE) approach and exploring different spin-labeling positions within RNA, we were able to track the location and determine the orientation of the RNA in the rS1–D34 bound form. Our investigations show that paramagnetically labeled RNAs, spiked into unmodified RNA, can be used as a molecular ruler to provide structural information on protein-RNA complexes. The dynamic interaction occurs on a defined binding groove spanning both domains with identical β2-β3-β5 interfaces. Evidently, the 3′-ends of the cis-acting RNAs are positioned in the direction of the N-terminus of the rS1 protein, thus towards the 30S binding site and adopt a conformation required for translation initiation.     

Three-dimensional homonuclear NOESY-TOCSY of an intramolecular pyrimidine.purine.pyrimidine DNA triplex containing a central G.TA triple: nonexchangeable proton assignments and structural implications.

Two- and three-dimensional homonuclear 1H NMR spectroscopic techniques have been applied to obtain nearly complete nonexchangeable proton assignments for a 31-residue intramolecular pyrimidine.purine.pyrimidine DNA triplex containing a central G.TA triple in D2O. An assignment strategy for obtaining resonance assignments for DNA protons from a 3D NOESY-TOCSY spectrum is proposed. The strategy utilizes the H1'/H5 omega 3 planes and relies on the recognition of cross-peak patterns for obtaining both intraresidue as well as sequential assignments. On the basis of the cross-peaks observed in the 2D and 3D spectra, a few structural features of the triplex have been delineated qualitatively. All three strands of the triplex adopt a right-handed helical conformation, and, despite the introduction of a central purine guanosine, there is no evidence for major structural distortions in the protonated third strand on the basis of a qualitative interpretation of NMR data. Several interstrand contacts between the purine and the Hoogsteen pyrimidine strands are observed which define the relative orientation of the bases and sugars in these two strands. The presence of strong NOEs between the methyl protons of thymine and the H1' proton of guanosine defines the preferred base-pairing alignment of guanosine at the G.TA triple site. The general approaches illustrated in this study extend the range of DNA molecules accessible for detailed structural investigation by high-resolution NMR spectroscopy.     

Spectroscopic properties and helical stabilities of 25-nt parallel-stranded linear DNA duplexes

DNA strands with appropriate sequences of dA and dT can form a stable duplex in which the two strands adopt a parallel (ps) instead of the conventional antiparallel (aps) orientation. Four 25-nt dA.dT-containing deoxyoligonucleotides (D1-4) were synthesized. D1 has the sequence 5'-dA10TA2T4A3TAT3-3'. Viewed with the same polarity, D2, D3, and D4 are the complement, inverted complement, and inverse of D1, respectively. The two combinations D1.D3 and D2.D4 form conventional antiparallel duplexes (aps-D1.D3, aps-D2.D4). D1.D2 and D3.D4, however, constitute stable parallel-stranded duplexes (ps-D1.D2, ps-D3.D4), as established by various criteria including the following: (i) The electrophoretic mobilities of ps-D1.D2 and ps-D3.D4 are similar to those of the antiparallel-stranded duplexes. (ii) The ultraviolet absorption and circular dichroism spectra of the ps duplexes are indicative of a base-paired structure, but differ systematically from those of the aps helices. (iii) Similar salt-dependent thermal transitions are observed for the four duplexes, but the melting temperatures of the ps molecules are lower by 13-18 degrees C.     

A parallel stranded linear DNA duplex incorporating dG.dC base pairs

DNA oligonucleotides with appropriately designed complementary sequences can form a duplex in which the two strands are paired in a parallel orientation and not in the conventional antiparallel double helix of B-DNA. All parallel stranded (ps) molecules reported to date have consisted exclusively of dA.dT base pairs. We have substituted four dA.dT base pairs of a 25-nt parallel stranded linear duplex (ps-D1.D2) with dG.dC base pairs. The two strands still adopt a duplex structure with the characteristic spectroscopic properties of the ps conformation but with a reduced thermodynamic stability. Thus, the melting temperature of the ps duplex with four dG.dC base pairs (ps-D5.D6) is 10-16 degrees C lower and the van't Hoff enthalpy difference delta HvH for the helix-coil transition is reduced by 20% (in NaCl) and 10% (in MgCl2) compared to that of ps-D1.D2. Based on energy minimizations of a ps-[d(T5GA5).d(A5CT5)] duplex using force field calculations we propose a model for the conformation of a trans dG.dC base pair in a ps helix.     

Assessment of dopamine D₁ receptor affinity and efficacy of three tetracyclic conformationally-restricted analogs of SKF38393

To assess the effect of conformational mobility on receptor activity, the β-phenyl substituent of dopamine D(1) agonist ligands of the phenylbenzazepine class, (±)-6,6a,7,8,9,13b-hexahydro-5H-benzo[d]naphtho[2,1-b]azepine-11,12-diol (8), and its oxygen and sulfur bioisosteres 9 and 10, respectively, were synthesized as conformationally-restricted analogs of SKF38393, a dopamine D(1)-selective partial agonist. Compounds trans-8b, 9, and 10 showed binding affinity comparable to that of SKF38393, but functionally, they displayed only very weak agonist activity. These results suggest that the conformationally-restricted structure of the analogs cannot adopt a binding orientation that is necessary for agonist activity.     

Cooperativity of carbohydrate moiety orientation and beta-turn stability is determined by intramolecular hydrogen bonds in protected glycopeptide models

The 2,3,4,6-Tetra-O-acetyl-beta-D-gluco-, and beta-D-galactopyranosides, as well as approximately 4:1 anomeric mixtures of alpha- and beta-mannopyranosides of Boc-X-Y-NHCH3 dipeptides (X-Y = Pro-Ser, Pro-D-Ser, Val-Ser, Val-D-Ser, and Gly-Ser) have been synthesized. CD and ir spectroscopic studies were performed to characterize the conformation of the glycosylated peptide backbone and examine the possible formation of intrapeptide and glycopeptide intramolecular H-bonds. It was found that O-glycosylated peptides containing a D-serine residue are likely to adopt a type II beta-turn while those with the Pro-Ser or Val-Ser sequence feature a type I (III) beta-turn in solution. Glycosylation also increases the magnitude of the CD bands, characteristic of the given type of beta-turns, which can be interpreted as an indication of the stabilization of the folded backbone conformation. Infrared data showed that in nonpolar solutions the peracetyl glycopeptides adopt both single- and double H-bonded conformations whose ratio, in some cases, depends on the position at C-2' of the H-bond acceptor acetoxy group. These data suggest that five-, seven-, or ten-membered glyco-turns may play an important role in fixing the steric orientation of the carbohydrate antennae systems in glycoproteins.     

Dimerization of helical β-peptides in solution

Molecular simulations are used to examine the aggregation behavior of several β-peptides in explicit water. The particular peptides considered here adopt a helical, rodlike conformation in aqueous solution. Four distinct molecular sequences are considered. Earlier experimental studies have revealed the formation of ordered and disordered aggregates for such molecules, depending on sequence. The simulations reported here, which are conducted by resorting to metadynamics techniques, lead to free energy surfaces for dimerization of the peptides in water as a function of separation and relative orientation. Such surfaces are used to identify the molecular origins for the behaviors observed in the experiments.     

猫视皮层细胞对运动光棒刺激的反应特性的定量分析

数据分析方法的不完善影响了关于细胞视觉反应特性研究结果的可靠性.本工作在分离视觉反应中方向和取向选择性因素的基础上.采用“积分平均”的手段.且将取向选择性理解为“强度随取向的变化率”,得到一种较为准确全面地表现细胞有关反应特性的定量分析法,可以体现出一些以往各方法无法反映出的差别,我们用此方法分析了猫视皮层细胞对运动光棒刺激的反应特性及其相关性.发现取向选择性因素对总反应的贡献较方向选择性大;反应强度,方向选择性和取向选择性三者间存在着弱相关.最优方向、最优取向及方向选择性最强的方向三者间有一定的偏离.     

Three-dimensional structure of the mammalian tachykinin peptide neurokinin A bound to lipid micelles

The solution structure of NKA, a decapeptide of mammalian origin, has been characterized by CD spectropolarimetry and 2D proton nuclear magnetic resonance (2D 1H-NMR) spectroscopy in both aqueous and membrane mimetic solvents. Unambiguous NMR assignments of protons have been made with the aid of correlation spectroscopy (DQF-COSY and TOCSY) experiments and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The distance constraints obtained from the NMR data have been utilized to generate a family of structures, which have been refined using restrained energy minimization and dynamics. These data show that in water NKA prefers to be in an extended chain conformation whereas a helical conformation is induced in the central core and the C-terminal region (D4-M10) of the peptide in the presence of perdeuterated dodecylphosphocholine (DPC) micelles, a membrane model system. Though less defined the N-terminus also displays some degree of order and a possible turn structure. The conformation adopted by NKA in the presence of DPC micelles represents a structural motif typical of neurokinin-2 selective agonists and is similar to that reported for eledoisin in hydrophobic environment.     

The glycine residue in cyclic lactam analogues of galanin(1-16)-NH2 is important for stabilizing an N-terminal helix

The neuropeptide galanin is a 29- or 30-residue peptide whose physiological functions are mediated by G-protein-coupled receptors. Galanin's agonist activity has been shown to be associated with the N-terminal sequence, galanin(1-16). Conformational investigations previously carried out on full-length galanin have, furthermore, indicated the presence of a helical conformation in the neuropeptide's N-terminal domain. Several cyclic lactam analogues of galanin(1-16)-NH2 were prepared in an attempt to stabilize an N-terminal helix in the peptide. Here we describe and compare the solution conformational properties of these analogues in the presence of SDS micelles as determined by NMR, CD, and fluorescence spectroscopy. Differences in CD spectral profiles were observed among the compounds that were studied. Both c[D4, K8]Gal(1-16)-NH2 and c[D4,K8]Gal(1-12)-NH2 adopted stable helical conformations in the micelle solution. On the basis of the analyses of their respective alpha H chemical shifts and NOE patterns, this helix was localized to the first 10 residues. The distance between the aromatic rings of Trp2 and Tyr9 in c[D4, K8]Gal(1-16)-NH2 was determined to be 10.8 +/- 3 A from fluorescence resonance energy transfer measurements. This interchromophore spacing was found to be more consistent with a helical structure than an extended one. Removal of the Gly1 residue in compounds c[D4,K8]Gal(1-16)-NH2 and c[D4, K8]Gal(1-12)-NH2 resulted in a loss of helical conformation and a concomitant reduction in binding potency at the GalR1 receptor but not at the GalR2 receptor. The nuclear Overhauser enhancements obtained for the Gly1 deficient analogues did, however, reveal the presence of nascent helical structures within the N-terminal sequence. Decreasing the ring structure size in c[D4, K8]Gal(1-16)-NH2 by replacing Lys8 with an ornithine residue or by changing the position of the single lysine residue from eight to seven was accompanied by a complete loss of helical structure and dramatically reduced receptor affinity. It is concluded from the data obtained for the series of cyclic galanin(1-16)-NH2 analogues that both the ring structure size and the presence of an N-terminal glycine residue are important for stabilizing an N-terminal helix in these compounds. However, although an N-terminal helix constitutes a predominant portion of the conformational ensemble for compounds c[D4,K8]Gal(1-16)-NH2 and c[D4, K8]Gal(1-12)-NH2, these peptides nevertheless are able to adopt other conformations in solution. Consequently, the correlation between the ability of the cyclic galanin analogues to adopt an N-terminal helix and bind to the GalR1 receptor may be considered as a working hypothesis.     

Orientation of a beta-hairpin antimicrobial peptide in lipid bilayers from two-dimensional dipolar chemical-shift correlation NMR

The orientation of a beta-sheet membrane peptide in lipid bilayers is determined, for the first time, using two-dimensional (2D) (15)N solid-state NMR. Retrocyclin-2 is a disulfide-stabilized cyclic beta-hairpin peptide with antibacterial and antiviral activities. We used 2D separated local field spectroscopy correlating (15)N-(1)H dipolar coupling with (15)N chemical shift to determine the orientation of multiply (15)N-labeled retrocyclin-2 in uniaxially aligned phosphocholine bilayers. Calculated 2D spectra exhibit characteristic resonance patterns that are sensitive to both the tilt of the beta-strand axis and the rotation of the beta-sheet plane from the bilayer normal and that yield resonance assignment without the need for singly labeled samples. Retrocyclin-2 adopts a transmembrane orientation in dilauroylphosphatidylcholine bilayers, with the strand axis tilted at 20 degrees +/- 10 degrees from the bilayer normal, but changes to a more in-plane orientation in thicker 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-choline (POPC) bilayers with a tilt angle of 65 degrees +/- 15 degrees . These indicate that hydrophobic mismatch regulates the peptide orientation. The 2D spectra are sensitive not only to the peptide orientation but also to its backbone (phi, psi) angles. Neither a bent hairpin conformation, which is populated in solution, nor an ideal beta-hairpin with uniform (phi, psi) angles and coplanar strands, agrees with the experimental spectrum. Thus, membrane binding orders the retrocyclin conformation by reducing the beta-sheet curvature but does not make it ideal. (31)P NMR spectra of lipid bilayers with different compositions indicate that retrocyclin-2 selectively disrupts the orientational order of anionic membranes while leaving zwitteronic membranes intact. These structural results provide insights into the mechanism of action of this beta-hairpin antimicrobial peptide.     

Validation of helical tilt angles in the solution NMR structure of the Z domain of Staphylococcal protein A by combined analysis of residual dipolar coupling and NOE data

Staphylococcal protein A (SpA) is a virulence factor from Staphylococcus aureus that is able to bind to immunoglobulins. The 3D structures of its immunoglobulin (Ig) binding domains have been extensively studied by NMR and X-ray crystallography, and are often used as model structures in developing de novo or ab initio strategies for predicting protein structure. These small three-helix-bundle structures, reported in free proteins or Ig-bound complexes, have been determined previously using medium- to high-resolution data. Although the location and relative orientation of the three helices in most of these published 3D domain structures are consistent, there are significant differences among the reported structures regarding the tilt angle of the first helix (helix 1). We have applied residual dipolar coupling data, together with nuclear Overhauser enhancement and scalar coupling data, in refining the NMR solution structure of an engineered IgG-binding domain (Z domain) of SpA. Our results demonstrate that the three helices are almost perfectly antiparallel in orientation, with the first helix tilting slightly away from the other two helices. We propose that this high-accuracy structure of the Z domain of SpA is a more suitable target for theoretical predictions of the free domain structure than previously published lower-accuracy structures of protein A domains.     

Molecular Conformation of the Full-Length Tumor Suppressor NF2/Merlin—A Small-Angle Neutron Scattering Study

The tumor suppressor protein Merlin inhibits cell proliferation upon establishing cell–cell contacts. Because Merlin has high level of sequence similarity to the Ezrin-Radixin-Moesin family of proteins, the structural model of Ezrin-Radixin-Moesin protein autoinhibition and cycling between closed/resting and open/active conformational states is often employed to explain Merlin function. However, recent biochemical studies suggest alternative molecular models of Merlin function. Here, we have determined the low-resolution molecular structure and binding activity of Merlin and a Merlin(S518D) mutant that mimics the inactivating phosphorylation at S518 using small-angle neutron scattering and binding experiments. Small-angle neutron scattering shows that, in solution, both Merlin and Merlin(S518D) adopt a closed conformation, but binding experiments indicate that a significant fraction of either Merlin or Merlin(S518D) is capable of binding to the target protein NHERF1. Upon binding to the phosphatidylinositol 4,5-bisphosphate lipid, the wild-type Merlin adopts a more open conformation than in solution, but Merlin(S518D) remains in a closed conformation. This study supports a rheostat model of Merlin in NHERF1 binding and contributes to resolving a controversy about the molecular conformation and binding activity of Merlin.     

A 1H NMR study of the solution conformation of the neuropeptide galanin.

The conformations of the neuropeptide galanin in water and trifluoroethanol solutions have been examined by 1H NMR spectroscopy. Analysis of two-dimensional NMR experiments enabled the assignment of virtually all the 1H resonances of galanin in trifluoroethanol solution and many of the 1H resonances in aqueous solution. Interpretation of the NMR data in structural terms suggests that in trifluoroethanol galanin is predominantly helical while in water it does not adopt a fixed conformation.     

NMR structure of a parallel-stranded DNA duplex at atomic resolution

DNA dodecamers have been designed with two cytosines on each end and intervening A and T stretches, such that the oligomers have fully complementary A:T base pairs when aligned in the parallel orientation. Spectroscopic (UV, CD and IR), NMR and molecular dynamics studies have shown that oligomers having the sequences d(CCATAATTTACC) and d(CCTATTAAATCC) form a parallel-stranded duplex when dissolved at 1:1 stoichiometry in aqueous solution. This is due to the C:C⁺ clamps on either end and extensive mismatches in the antiparallel orientation. The structure is stable at neutral and acidic pH. At higher temperatures, the duplex melts into single strands in a highly cooperative fashion. All adenine, cytosine and thymine nucleotides adopt the anti conformation with respect to the glycosidic bond. The A:T base pairs form reverse Watson–Crick base pairs. The duplex shows base stacking and NOEs between the base protons T(H6)/A(H8) and the sugar protons (H1′/H2′/H2″) of the preceding nucleotide, as has been observed in antiparallel duplexes. However, no NOEs are observed between base protons H2/H6/H8 of sequential nucleotides, though such NOEs are observed between T(CH₃) and A(H8). A three-dimensional structure of the parallel-stranded duplex at atomic resolution has been obtained using molecular dynamics simulations under NMR constraints. The simulated structures have torsional angles very similar to those found in B-DNA duplexes, but the base stacking and helicoid parameters are significantly different.