The Stabilizing Effects of O-glycosylation on the Secondary Structural Integrity of the Designed α-loop-α motif by Molecular Dynamics Simulations

Abstract

In this study, various 400 ps molecular dynamics simulations were conducted to determine the stabilizing effect of O-glycosylation on the secondary structural integrity of the design α-loop-α motif, which has the optimal loop length of 7 Gly residues (denoted as N-A₁₆G₇A₁₆-C). In general, O-glycosylation stabilizes the structural integrity of the model peptide regardless of the length and position of glycosylation sites because it decreases the opportunity for water molecules to compete for the intramolecular hydrogen bonds. The designed peptide exhibits the highest helicity when residues 11 and 31 are replaced with Ser residues followed by O-linked with 3 galactose residues, representing the “face-to-face” glycosylation near the loop. In this case, the loop exhibits an extended conformation and several new hydrogen bonds are observed between the main chain of the loop and the galactose residues, resulting in decreasing the fluctuation and increasing the stability of the entire peptide. When the glycosylation are made close to the loop, the secondary structural integrity of the α-loop-α motif increases with the number of galactose residues. In addition, “face- to-face” glycosylation increases the structural integrity of this motif to a greater extent than “back-to-back” glycosylation. However, when the glycosylation are created away from the loop and near the N- and C-termini, no general rule is found for the stabilizing effect.     

Carboxyl pKa Values and Acid Denaturation of BBL

The protein BBL undergoes structural transitions and acid denaturation between pH 1.2 and 8.0. Using NMR spectroscopy, we measured the pK_a values of all the carboxylic residues in this pH range. We employed ¹³C direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring pK_a values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pK_a values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed pK_a, and, using thermodynamic cycles, we could calculate their pK_as in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured ¹³C^α chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH 7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state.     

The 5′ Stem-Loop From Human U4 snRNA Adopts a Novel Conformation Stabilized by G-C and G-U Base Pairs

Abstract

¹H NMR and molecular modeling studies of the 5′ stem-loop from human U4 snRNA were undertaken to determine the conformation of this stem-loop that is essential for spliceosome formation and pre-mRNA splicing. Sixteen of the 35 nucleotides of this stem-loop are in the loop region and inspection of the loop sequence revealed no decomposition into elements of secondary structure commonly found in other RNA stem-loops. An analysis of possible base pairing interactions for this stem-loop using the methods of Zuker revealed the lowest energy secondary structure for the 16 nucleotide loop consisted of four base pairs at the base of a non-canonical tetraloop (UUUA). This shorter stem-loop was joined to the nine base pair stem by two A residues on the 5′ side and a single bulged A on the 3′ side. Both stems also had bulged A residues. ¹H NMR experiments performed on solutions of the 35mer stem-loop, the stem region, and the loop region confirmed the 35mer adopted this secondary structure in solution. A 3D molecular model of this structure consistent with the NMR data was generated to assist in visualization of this novel structure.     

Enhanced loop DNA folding induced by thymine-CH3 group contact and perpendicular guanine-thymine interaction

A remarkable stabilizing effect induced by T-CH₃ group and perpendicular guanine–thymine interactions in the DNA loop conformation has been demonstrated for the d(TTTG) loop structure using UV melting, high resolution NMR, distance geometry, and molecular dynamics studies. Contrary to the previously published d(TTCG) sequence that exhibits no specific inter-nucleotide interaction, we have found that d(TTTG), which differs only by one nucleotide with the d(TTCG) sequence (C7 T7), forms a rather stable and well-defined loop structure. Two characteristic structural features account for the stabilization of an otherwise flexible loop structure; the second loop T (T6) residue folds into the minor groove and engages in perpendicular interaction with the G8-NH₂, while the third loop T (T7) residue stacks well upon the closing T5G8 wobble base pair and exhibits good contacts with many of the loop T5 and T6 sugar protons, which may form a hydrophobic core in the loop region. The importance of the bulky T7-CH₃ was also proved by the UV melting study; while d(TTCG) hairpin exhibits a lower melting point (74.5°C ) than d(TTTG) hairpin (80.5°C ), d(TT^5–methylCG) hairpin resumes the same higher melting point (80°C ). Similarly, the fact that the melting temperature (74°C ) of d(TTTI) is lower than that of d(TTTG) indicates the critical role played by the G8-NH₂ group. Our structural studies of the d(TTTG) loop indicate that DNA and RNA use a different strategy to establish stable tertiary folds. Comparison with several other pyrimidine-rich loop hairpins suggests that different minor-groove folding modes exist for the folding thymidine residue.     

Solution structure of porcine pancreatic phospholipase A2 complexed with micelles and a competitive inhibitor

Summary The three-dimensional structure of porcine pancreatic PLA₂ (PLA₂), present in a 40 kDa ternary complex with micelles and a competitive inhibitor, has been determined using multidimensional heteronuclear NMR spectroscopy. The structure of the protein (124 residues) is based on 1854 constraints, comprising 1792 distance and 62 torsion angle constraints. A total of 18 structures was calculated using a combined approach of distance geometry and restrained molecular dynamics. The atomic rms distribution about the mean coordinate positions for residues 1–62 and 72–124 is 0.75±0.09 Å for the backbone atoms and 1.14±0.10 Å for all atoms. The rms difference between the averaged minimized NMR structures of the free PLA₂ and PLA₂ in the ternary complex is 3.5 Å for the backbone atoms and 4.0 Å for all atoms. Large differences occur for the calcium-binding loop and the surface loop from residues 62 through 72. The most important difference is found for the first three residues of the N-terminal -helix. Whereas free in solution Ala¹, Leu² and Trp³ are disordered, with the -amino group of Ala¹ pointing out into the solvent, in the ternary complex these residues have an -helical conformation with the -amino group buried inside the protein. As a consequence, the important conserved hydrogen bonding network which is also seen in the crystal structures is present only in the ternary complex, but not in free PLA₂. Thus, the NMR structure of the N-terminal region (as well as the calcium-binding loop and the surface loop) of PLA₂ in the ternary complex resembles that of the crystal structure. Comparison of the NMR structures of the free enzyme and the enzyme in the ternary complex indicates that conformational changes play a role in the interfacial activation of PLA₂.     

Role of connecting loop I in catalysis and allosteric regulation of human glucokinase

Glucokinase (GCK, hexokinase IV) is a monomeric enzyme with a single glucose binding site that displays steady‐state kinetic cooperativity, a functional characteristic that affords allosteric regulation of GCK activity. Structural evidence suggests that connecting loop I, comprised of residues 47–71, facilitates cooperativity by dictating the rate and scope of motions between the large and small domains of GCK. Here we investigate the impact of varying the length and amino acid sequence of connecting loop I upon GCK cooperativity. We find that sequential, single amino acid deletions from the C‐terminus of connecting loop I cause systematic decreases in cooperativity. Deleting up to two loop residues leaves the k_cat value unchanged; however, removing three or more residues reduces k_cat by 1000‐fold. In contrast, the glucose K_0.5 and K_D values are unaffected by shortening the connecting loop by up to six residues. Substituting alanine or glycine for proline‐66, which adopts a cis conformation in some GCK crystal structures, does not alter cooperativity, indicating that cis/trans isomerization of this loop residue does not govern slow conformational reorganizations linked to hysteresis. Replacing connecting loop I with the corresponding loop sequence from the catalytic domain of the noncooperative isozyme human hexokinase I (HK‐I) eliminates cooperativity without impacting the k_cat and glucose K_0.5 values. Our results indicate that catalytic turnover requires a minimal length of connecting loop I, whereas the loop has little impact upon the binding affinity of GCK for glucose. We propose a model in which the primary structure of connecting loop I affects cooperativity by influencing conformational dynamics, without altering the equilibrium distribution of GCK conformations.     

Crystal structure of Bence Jones protein rhe (3 A) and its unique domain-domain association.

The crystal structure of protein Rhe, a lambda type V_L dimer, has been determined at a resolution of 3 Å by the method of multiple isomorphous replacement supplemented with anomalous scattering data. A crystallographic sequence was assigned from an interpretation of the electron density map in an optical comparator and is compared with a chemically determined partial amino acid sequence. The monomeric unit of Rhe, as determined crystallographically, contains 113 amino acids, 110 belonging to the variable region and three belonging to the constant segment of a light chain. The single polypeptide chain constituting the monomer forms a nine-stranded β-barrel characteristic of V domains. The β-pleated sheet surrounds an ellipsoidally shaped interior core of approximately 10 Å × 15 Å × 25 Å in size. The monomers that are related by the crystallographic dyad are held together as dimers by interdomain hydrogen bonds and hydrophobic interactions. At one end of the dimer is an opening which is lined exclusively by residues from the hypervariable regions.A comparison of Rhe with Rei, a kappa type V_L dimer (Epp et al., 1975), revealed that monomers of Rhe and Rei dimerized differently. Their respective dyad and pseudodyad of dimerization are not the same, and this causes a variation in the overall steric arrangement of the hypervariable regions in the two cavities. In adition a dissimilarity was observed in the non-hypervariable segment linking the first and second hypervariable regions. This segment is in the form of a loop and it includes most of the residues participating in the interdomain interactions stabilizing dimer formation in both proteins and these loop positions differ by as much as 7 Å. Our results also show that there is a good correlation between the dissimilarity of the loop position and the difference in the domain-association. Our preliminary analysis indicates that the positions of the corresponding non-hypervariable loops in V domains may be determined in part by the residues in the hypervariable regions.Accordingly, the three-dimensional structure of Rhe suggests that this nonhypervariable loop in V_L and its counterpart in V_H may have an important biological function in antibody specificity and variability by virtue of their influence over the architecture of the complementarity site.     

Diverse residues of intracellular loop 5 of the Na+/H+ exchanger modulate proton sensing,expression, activity and targeting

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH (pH_i) by removing a single intracellular proton in exchange for one extracellular sodium ion. It is involved in cardiac hypertrophy and ischemia reperfusion damage to the heart and elevation of its activity is a trigger for breast cancer metastasis. NHE1 has an extensive 500 amino acid N-terminal membrane domain that mediates transport and consists of 12 transmembrane segments connected by intracellular and extracellular loops. Intracellular loops are hypothesized to modulate the sensitivity to pH_i. In this study, we characterized the structure and function of intracellular loop 5 (IL5), specifically amino acids 431–443. Mutation of eleven residues to alanine caused partial or nearly complete inhibition of transport; notably, mutation of residues L432, T433, I436, N437, R440 and K443 demonstrated these residues had critical roles in NHE1 function independent of effects on targeting or expression. The nuclear magnetic resonance (NMR) solution spectra of the IL5 peptide in a membrane mimetic sodium dodecyl sulfate solution revealed that IL5 has a stable three-dimensional structure with substantial alpha helical character. NMR chemical shifts indicated that K438 was in close proximity with W434. Overall, our results show that IL5 is a critical, intracellular loop with a propensity to form an alpha helix, and many residues of this intracellular loop are critical to proton sensing and ion transport.     

The roles of the conserved tyrosine in the β2-α2 loop of the prion protein

ABSTRACT

Prions cause neurodegenerative diseases for which no cure exists. Despite decades of research activities the function of the prion protein (PrP) in mammalians is not known. Moreover, little is known on the molecular mechanisms of the self-assembly of the PrP from its monomeric state (cellular PrP, PrP^C) to the multimeric state. The latter state includes the toxic species (scrapie PrP, PrP^Sc) knowledge of which would facilitate the development of drugs against prion diseases. Here we analyze the role of a tyrosine residue (Y169) which is strictly conserved in mammalian PrPs. Nuclear magnetic resonance (NMR) spectroscopy studies of many mammalian PrP^C proteins have provided evidence of a conformational equilibrium between a 3₁₀-helical turn and a type I β turn conformation in the β2-α2 loop (residues 165–175). In vitro cell-free experiments of the seeded conversion of PrP^C indicate that non-aromatic residues at position 169 reduce the formation of proteinase K-resistant PrP. Recent molecular dynamics (MD) simulations of monomeric PrP and several single-point mutants show that Y169 stabilizes the 3₁₀-helical turn conformation more than single-point mutants at position 169 or residues in contact with it. In the 3₁₀-helical turn conformation the hydrophobic and aggregation-prone segment 169-YSNQNNF-175 is buried and thus not-available for self-assembly. From the combined analysis of simulation and experimental results it emerges that Y169 is an aggregation gatekeeper with a twofold role. Mutations related to 3 human prion diseases are interpreted on the basis of the gatekeeper role in the monomeric state. Another potential role of the Y169 side chain is the stabilization of the ordered aggregates, i.e., reduction of frangibility of filamentous protofibrils and fibrils, which is likely to reduce the generation of toxic species.     

Probing excited states and activation energy for the integral membrane protein phospholamban by NMR CPMG relaxation dispersion experiments

Phospholamban (PLN) is a dynamic single-pass membrane protein that inhibits the flow of Ca²⁺ ions into the sarcoplasmic reticulum (SR) of heart muscle by directly binding to and inhibiting the SR Ca²⁺ATPase (SERCA). The PLN monomer is the functionally active form that exists in equilibrium between ordered (T state) and disordered (R state) states. While the T state has been fully characterized using a hybrid solution/solid-state NMR approach, the R state structure has not been fully portrayed. It has, however, been detected by both NMR and EPR experiments in detergent micelles and lipid bilayers. In this work, we quantitatively probed the μs to ms dynamics of the PLN excited states by observing the T state in DPC micelles using CPMG relaxation dispersion NMR spectroscopy under functional conditions for SERCA. The ¹⁵N backbone and ¹³C_δ1 Ile-methyl dispersion curves were fit using a two-state equilibrium model, and indicate that residues within domain Ia (residues 1-16), the loop (17-22), and domain Ib (23-30) of PLN undergo μs-ms dynamics (k_ex = 6100 ±800 s^- 1 at 17 °C). We measured k_ex at additional temperatures, which allowed for a calculation of activation energy equal to ∼ 5 kcal/mol. This energy barrier probably does not correspond to the detachment of the amphipathic domain Ia, but rather the energy needed to unwind domain Ib on the membrane surface, likely an important mechanism by which PLN converts between high and low affinity states for its binding partners.     

Characterization of the Structure and Melting Behavior of the Loop I fragment of ColE1 RNA I

Abstract

We have synthesized two RNA fragments: a 42-mer corresponding to the full loop I sequence of the loop I region of ColE1 antisense RNA (RNA I), plus three additional Gs at the 5′-end, and a 31-mer which has 11 5′-end nucleotides (G(-2)-U9) deleted. The secondary structure of the 42-mer, deduced from one- and two-dimensional NMR spectra, consists of a stem of 11 base-pairs which contains a U-U base-pair and a bulged C base, a 7 nucleotide loop, and a single-stranded 5′ end of 12 nucleotides. The UV-melting study of the 42-mer further revealed a multi-step melting behavior with transition temperatures 32°C and 71°C clearly discernible. In conjunction with NMR melting study the major transition at 71°C is assigned to the overall melting of the stem region and the 32°C transition is assigned to the opening of the loop region. The deduced secondary structure agrees with that proposed for the intact RNA I and provides structural bases for understanding the specificity of RNase E.     

Loop Folds in Proteins and Evolutionary Conservation of Folding Nuclei

Abstract

We show that loops of close contacts involving hydrophobic residues are important in protein folding. Contrary to Berezovsky and Trifonov (J. Biomol. Struct. Dyn. 20, 5–6, 2002) the loops important in protein folding usually are much larger in size than 23–31 residues, being instead comparable to the size of the protein for single domain proteins. Additionally what is important are not single loop contacts, but a highly interconnected network of such loop contacts, which provides extra stability to a protein fold and which leads to their conservation in evolution.     

Molecular Mechanics and Dynamics of an Abasic Frameshift in DNA and Comparison to NMR Data

Abstract

In a previous publication (Ph. Cuniasse, L.C. Sowers, R. Eritja, B. Kaplan, M.F. Goodman, J.A.H. Cognet, M. Le Bret, W. Guschlbauer and G.V. Fazakerley, Biochemistry 28, 2018 (1989), we determined by two dimensional NMR studies and molecular mechanics calculations the three-dimensional structure of a non-selfcomplementary oligonucleotide:

5′d(C1 P1 G2 P2 G3 P3 dr4 P4 G5 P5 G6 P6 C7)3′

3′d(G13P12C12PllCll P10 C10 P9 C9 P8 G8)5′

where dr, at the center of the first strand, is a model abasic site. In order to explain all the results arising from NMR measurements, we found that an equilibrium between two conformations was necessary. These conformations differ mainly by the sugar pucker of G5 which is C2′ endo or C3′ endo. The latter is stabilized by addition of counterions between phosphate residues P3 and P4.

In this paper, we have constructed systematically, all possible structures as a function of torsion angles delta of dr4 and of G5 by molecular mechanics in the presence or absence of counterions. Since these conformations were not forced with NMR distance measurements, this method allows detailed comparisons between all possible conformations and NMR data. Maps of contour lines of the potential energy, of fits to NMR distance measurements, and of helical twist as a function of torsion angles delta of dr4 and of G5 unravel the difficulties associated with the study of the G5 sugar pucker conformation equilibrium.

Sugar puckers and proton distances are very sensitive criteria to monitor molecular dynamics. Relying on these experimental criteria, we have tested many molecular dynamics preparation phases and we propose a new warm-up and equilibration procedure for molecular dynamics. Thus we show with a 290 ps molecular dynamic run that G5 is in conformational equilibrium and that all NMR data are well reproduced.     

Crystal structure of the crenarchaeal conserved chromatin protein Cren7 and double-stranded DNA complex

Cren7 is a crenarchaeal conserved chromatin protein discovered recently. To explore the mechanism of the DNA packaging in Crenarchaeota, the crystal structure of Cren7–GCGATCGC complex has been determined and refined at 1.6 Å resolution. Cren7 kinks the dsDNA sharply similar to Sul7d, another chromatin protein existing only in Sulfolobales, which reveals that the “bending and unwinding” compacting mechanism is conserved in Crenarchaeota. Significant structural differences are revealed by comparing both protein–dsDNA complexes. The kinked sites on the same dsDNA in the complexes with Sul7d and Cren7 show one base pair shift. For Cren7, fewer charged residues in the β‐barrel structural region bind to DNA, and additionally, the flexible loop L_β3β4 is also involved in the binding. Electrophoretic mobility shift assays indicate that loop L_β3β4 is essential for DNA‐binding of Cren7. These differences provide insight into the functional difference of both chromatin proteins, suggesting that Cren7 may be more regulative than Sul7d in the DNA‐binding affinity by the methylation in the flexible loop L_β3β4 in vivo.     

Conformational and Model-Building Studies of the Hairpin Form of the Mismatched DNA Octamer d(m5C-G-m5C-G-T-G-m5C-G)

Abstract

The hairpin form of the mismatched octamer d(m⁵C-G-m⁵C-G-T-G-m⁵C-G) was studied by means of NMR spectroscopy. In a companion study it is shown that the hairpin form of this DNA fragment consists of a structure with a stem of three Watson-Crick-type base pairs and a loop consisting of only two nucleotides. The non-exchangeable proton resonances were assigned by means of two-dimensional correlation spectroscopy and two-dimensional nuclear Overhauser effect spectroscopy. Proton-proton coupling constants were used for the conformational analysis of the deoxyribose ring and for some of the backbone torsion angles. From the two-dimensional NMR spectra and the coupling-constant analysis it is concluded that: (i) the stem of the hairpin exhibits B-DNA characteristics; (ii) the sugar rings are not conformationally pure, but display a certain amount of conformational flexibility; (iii) the stacking interaction in the stem of the hairpin is elongated from the 3′-side in a more or less regular fashion with the two loop nucleotides; (iv) at the 5′-side of the stem a stacking discontinuity occurs between the stem and the loop; (v) at the 5′-side of the stem the loop is closed by means of a sharp backbone turn which involves unusual γ^t and β⁺ torsion angles in residue dG(6).

The NMR results led to the construction of a hairpin-loop model which was energy-minimized by means of a molecular-mechanics program. The results clearly show that a DNA hairpin-loop structure in which the loop consists of only two nucleotides bridging the minor groove in a straightforward fashion, (i) causes no undue steric strain, and (ii) involves well-known conformational principles throughout the course of the backbone.

The hairpin form of the title compound is compared with the hairpin form of d(A-T-C-C-T- A-T₄-T-A-G-G-A-T), in which the central -T₄- part forms a loop of four nucleotides. Both models display similarities as far as stacking interactions are concerned.     

A Protoberberine Derivative HWY336 Selectively Inhibits MKK4 and MKK7 in Mammalian Cells: The Importance of Activation Loop on Selectivity

A protoberberine derivative library was used to search for selective inhibitors against kinases of the mitogen-activated protein kinase (MAPK) cascades in mammalian cells. Among kinases in mammalian MAPK pathways, we identified a compound (HWY336) that selectively inhibits kinase activity of mitogen-activated protein kinase kinase 4 and 7 (MKK4 and MKK7). The IC₅₀ of HWY336 was 6 µM for MKK4 and 10 µM for MKK7 in vitro. HWY336 bound to both kinases reversibly via noncovalent interactions, and inhibited their activity by interfering with access of a protein substrate to its binding site. The binding affinity of HWY336 to MKK4 was measured by surface plasmon resonance to determine a dissociation constant (K_d) of 3.2 µM. When mammalian cells were treated with HWY336, MKK4 and MKK7 were selectively inhibited, resulting in inhibition of c-Jun NH₂-terminal protein kinases in vivo. The structural model of HWY336 bound to either MKK4 or MKK7 predicted that HWY336 was docked to the activation loop, which is adjacent to the substrate binding site. This model suggested the importance of the activation loop of MKKs in HWY336 selectivity. We verified this model by mutating three critical residues within this loop of MKK4 to the corresponding residues in MKK3. The mutant MKK4 displayed similar kinase activity as wild-type kinase, but its activity was not inhibited by HWY336 compared to wild-type MKK4. We propose that the specific association of HWY336 to the activation loop of MKK4/MKK7 is responsible for its selective inhibition.     

Toward the molecular basis of inherited prion diseases: NMR structure of the human prion protein with V210I mutation

The development of transmissible spongiform encephalopathies (TSEs) is associated with the conversion of the cellular prion protein (PrP^C) into a misfolded, pathogenic isoform (PrP^Sc). Spontaneous generation of PrP^Sc in inherited forms of disease is caused by mutations in gene coding for PrP (PRNP). In this work, we describe the NMR solution-state structure of the truncated recombinant human PrP (HuPrP) carrying the pathological V210I mutation linked to genetic Creutzfeldt-Jakob disease. The three-dimensional structure of V210I mutant consists of an unstructured N-terminal part (residues 90-124) and a well-defined C-terminal domain (residues 125-228). The C-terminal domain contains three α-helices (residues 144-156, 170-194 and 200-228) and a short antiparallel β-sheet (residues 129-130 and 162-163). Comparison with the structure of the wild-type HuPrP revealed that although two structures share similar global architecture, mutation introduces some local structural differences. The observed variations are mostly clustered in the α₂-α₃ inter-helical interface and in the β₂-α₂ loop region. Introduction of bulkier Ile at position 210 induces reorientations of several residues that are part of hydrophobic core, thus influencing α₂-α₃ inter-helical interactions. Another important structural feature involves the alteration of conformation of the β₂-α₂ loop region and the subsequent exposure of hydrophobic cluster to solvent, which facilitates intermolecular interactions involved in spontaneous generation of PrP^Sc. The NMR structure of V210I mutant offers new clues about the earliest events of the pathogenic conversion process that could be used for the development of antiprion drugs.     

Chemically accurate protein structures: Validation of protein NMR structures by comparison of measured and predicted pK a values

A new method is presented for evaluating the quality of protein structures obtained by NMR. This method exploits the dependence between measurable chemical properties of a protein, namely pK _a values of acidic residues, and protein structure. The accurate and fast empirical computational method employed by the PROPKA program () allows the user to test the ability of a given structure to reproduce known pK _a values, which in turn can be used as a criterion for the selection of more accurate structures. We demonstrate the feasibility of this novel idea for a series of proteins for which both␣NMR and X-ray structures, as well as pK _a values of all ionizable residues, have been determined. For the 17 NMR ensembles used in this study, this criterion is shown effective in the elimination of a large number of NMR structure ensemble members.     

An NMR Study of the Polymorphous Behavior of the Mismatched DNA Octamer d(m5C-G-m5C-G-T-G-m5C-G) in Solution. The B,Z, and Hairpin Forms

Abstract

The polymorphism exhibited by the mismatched octamer d(m ⁵C-G-m⁵C-G-T-G-m⁵C-G), as a function of the temperature, DNA concentration and ionic strength, was investigated by means of NMR spectroscopy.

It is shown that this partly self-complementary DNA fragment, under conditions of low DNA concentration (0.4 mM) and low ionic strength, exclusively prefers to adopt a monomeric hairpin form, which consists of a stem of three Watson-Crick-type base pairs and a loop of only two residues. This in striking contrast with earlier intimations in literature, which postulated that in oligonucleotides loop formations containing only two residues are sterically impossible. Moreover, the hairpin form displays an unusual stability in comparison with previously reported hairpins. AT_m of 332 K and a ΔH° of—130 kj · mol^?1 were calculated for the hairpin to random coil transition.

At high DNA concentration (8 mM)and/or upon the addition of sodium chloride the hairpin form occurs in slow exchange with a B-DNA dimer structure (approximately 20% at 270 K, no added salt), which comprises two central GxT-mismatched base pairs with the bases as major tautomers.

At higher ionic strength (> 100 mM NaCI), or upon the addition of methanol, a third species appears, which is in slow exchange with both the B dimer and the hairpin form. This third species could be identified with a Z DNA form, comprising two GxT mismatches with the bases as major tautomers, with the guanine bases syn and the cytosine and thymine bases anti.     

Evidence That Calmodulin Binding to the Dopamine D2 Receptor Enhances Receptor Signaling

The Ca²⁺ sensor calmodulin (CaM) regulates numerous proteins involved in G protein-coupled receptor (GPCR) signaling. CaM binds directly to some GPCRs, including the dopamine D₂ receptor. We confirmed that the third intracellular loop of the D₂ receptor is a direct contact point for CaM binding using coimmunoprecipitation and a polyHis pull-down assay, and we determined that the D₂-like receptor agonist 7-OH-DPAT increased the colocalization of the D₂ receptor and endogenous CaM in both 293 cells and in primary neostriatal cultures. The N-terminal three or four residues of D₂-IC3 were required for the binding of CaM; mutation of three of these residues in the full-length receptor (I210C/K211C/I212C) decreased the coprecipitation of the D₂ receptor and CaM and also significantly decreased D₂ receptor signaling, without altering the coupling of the receptor to G proteins. Taken together, these findings suggest that binding of CaM to the dopamine D₂ receptor enhances D₂ receptor signaling.