An on/off resonance rotating frame relaxation experiment to monitor millisecond to microsecond timescale dynamics

Rotating frame relaxation experiments in proteins are used to study slow motions on the microsecond to millisecond timescale. An on/off resonance rotating frame relaxation experiment (R(1)(rho)) has been developed that incorporates adiabatic rotations into a R(1)(rho)-R(1) constant relaxation time experiment with weak radio frequency field strengths in order to effectively lock the magnetization over a wide range of (15)N frequencies. The new pulse sequence allows the measurement of a wide range of chemical exchange timescales on the order of 1.0 to 0.05 ms over an asymmetric bandwidth from +1.7omega(l) to -0.5omega(l) in a single experiment. A total bandwidth of +/-l.7omega(l) is obtained by performing the experiment a second time with a reversed adiabatic rotation.     

Ribonuclease activity associated with human eosinophil-derived neurotoxin and eosinophil cationic protein

The eosinophil granule contains a series of basic proteins, including major basic protein, eosinophil peroxidase, eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP). Both EDN and ECP are neurotoxins and helminthotoxins. Comparison of the partial N-terminal amino acid sequences of EDN and ECP showed 67% identity; surprisingly, they also showed structural homology to pancreatic ribonuclease (RNase). Therefore, we determined whether EDN and ECP possess RNase enzymatic activity. By spectrophotometric assay of acid soluble nucleotides formed from yeast RNA, purified EDN showed RNase activity similar to bovine pancreatic RNase, whereas ECP was 50 to 100 times less active. The RNase activity associated with ECP was not significantly inhibited after exposure of ECP to polyclonal or monoclonal antibody to EDN. These results indicate that EDN and ECP both possess RNase activity, the RNase activity of EDN and ECP is specific, and EDN and ECP have maintained not only structural but also functional homology to pancreatic RNase.     

Sequence-specific 1H and 15N resonance assignments and secondary structure of GDP-bound human c-Ha-Ras protein in solution

Summary All the backbone ¹H and ¹⁵N magnetic resonances (except for Pro residues) of the GDP-bound form of a truncated human c-Ha-ras proto-oncogene product (171 amino acid residues, the Ras protein) were assigned by ¹⁵N-edited two-dimensional NMR experiments on selectively ¹⁵N-labeled Ras proteins in combination with three-dimensional NMR experiments on the uniformly ¹⁵N-labeled protein. The sequence-specific assignments were made on the basis of the nuclear Overhauser effect (NOE) connectivities of amide protons with preceding amide and/or Cprotons. In addition to sequential NOEs, vicinal spin coupling constants for amide protons and C protons and deuterium exchange rates of amide protons were used to characterize the secondary structure of the GDP-bound Ras protein; six strands and five helices were identified and the topology of these elements was determined. The secondary structure of the Ras protein in solution was mainly consistent with that in crystal as determined by X-ray analyses. The deuterium exchange rates of amide protons were examined to elucidate the dynamic properties of the secondary structure elements of the Ras protein in solution. In solution, the -sheet structure in the Ras protein is rigid, while the second helix (A66-R73) is much more flexible, and the first and fifth helices (S17-124 and V152-L171) are more rigid than other helices. Secondary structure elements at or near the ends of the effector-region loop were found to be much more flexible in solution than in the crystalline state.     

Sequence-specific 1H NMR assignments and secondary structure in the sea anemone polypeptide Stichodactyla helianthus neurotoxin I

Sequence-specific assignments are reported for the 500-MHz 1H nuclear magnetic resonance (NMR) spectrum of the 48-residue polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus (Sh I). Spin systems were first identified by using two-dimensional relayed or multiple quantum filtered correlation spectroscopy, double quantum spectroscopy, and spin lock experiments. Specific resonance assignments were then obtained from nuclear Overhauser enhancement (NOE) connectivities between protons from residues adjacent in the amino acid sequence. Of a total of 265 potentially observable resonances, 248 (i.e., 94%) were assigned, arising from 39 completely and 9 partially assigned amino acid spin systems. The secondary structure of Sh I was defined on the basis of the pattern of sequential NOE connectivities, NOEs between protons on separate strands of the polypeptide backbone, and backbone amide exchange rates. Sh I contains a four-stranded antiparallel beta-sheet encompassing residues 1-5, 16-24, 30-33, and 40-46, with a beta-bulge at residues 17 and 18 and a reverse turn, probably a type II beta-turn, involving residues 27-30. No evidence of alpha-helical structure was found.     

Mutational analysis of the complex of human RNase inhibitor and human eosinophil-derived neurotoxin (RNase 2)

RNase inhibitor (RI) binds diverse proteins in the pancreatic RNase superfamily with extremely high avidity. Previous studies showed that tight binding of RNase A and angiogenin (Ang) is achieved primarily through interactions of hot spot residues in the 434-460 C-terminal segment of RI with the enzymatic active site; Asp435 of RI forms key hydrogen bonds with the catalytic lysine in both complexes, whereas the other contacts are largely distinctive. Here we have investigated the structural basis for recognition of a third ligand, eosinophil-derived neurotoxin (EDN), by single-site and multisite mutagenesis. Surprisingly, Ala replacement of Asp435 decreases affinity for EDN only by 14-fold, as compared to the several hundred-fold decreases with RNase A and Ang, and individual mutations of three other hot spot residues-Tyr434, Tyr437, and Ser460-have essentially no effect. Ala substitutions of nine additional residues, selected by examining a computational model of the RI.EDN complex, also have no marked impact. Overall, the losses in affinity for the single-residue variants examined account for only approximately 25% of the free energy of binding for the complex. However, multisite mutagenesis of RI reveals strong superadditivity of mutational effects, indicating that part of this shortfall reflects negative cooperativity. Replacement of Tyr434 together with Asp435 or Tyr437 increases K(i) by 540- and 290-fold, respectively. Thus, the C-terminal region of RI again plays an important role in ligand recognition, although probably smaller than for binding RNase A and Ang. Simultaneous substitutions of three neighboring tryptophans (261, 263, and 318) on RI attenuate affinity even more dramatically (by 4900-fold), indicating that the interactions of this RI region also contribute a considerable amount of the binding energy for the EDN complex. These findings highlight the potential importance of cooperativity in protein-protein interactions and the consequent limitations of single-site mutagenesis for assessing interface energetics.     

Conformational dynamics of full-length inducible human Hsp70 derived from microsecond molecular dynamics simulations in explicit solvent

Human 70?kDa heat shock protein (hHsp70) is an ATP-dependent chaperone and is currently an important target for developing new drugs in cancer therapy. Knowledge of the conformations of hHsp70 is central to understand the interactions between its nucleotide-binding domain (NBD) and substrate-binding domain (SBD) and is a prerequisite to design inhibitors. The conformations of ADP-bound (or nucleotide-free) hHsp70 and ATP-bound hHsp70 was investigated by using unbiased all-atom molecular dynamics (MD) simulations of homology models of hHsp70 in explicit solvent on a timescale of .5 and 2.7 μs, respectively. The conformational heterogeneity of hHsp70 was analyzed by computing effective free-energy landscapes (FELs) and distance distribution between selected pair of residues. These theoretical data were compared with those extracted from single-molecule Förster resonance energy transfer (FRET) experiments and to small-angle X-ray scattering (SAXS) data of Hsp70 homologs. The distance between a pair of residues in FRET is systematically larger than the distance computed in MD which is interpreted as an effect of the size and of the dynamics of the fluorescent probes. The origin of the conformational heterogeneity of hHsp70 in the ATP-bound state is due to different binding modes of the helix B of the SBD onto the NBD. In the ADP-bound (or nucleotide-free) state, it arises from the different closed conformations of the SBD and from the different positions of the SBD relative to the NBD. In each nucleotide-binding state, Hsp70 is better represented by an ensemble of conformations on a μs timescale corresponding to different local minima of the FEL.

An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:30     

Sequence-specific 1H-NMR assignments and folding topology of human CD59.

CD59 is a recently discovered cell-surface glycoprotein that restricts lysis by homologous complement and has limited sequence similarity to snake venom neurotoxins. This paper describes the first results of a two-dimensional NMR study of CD59 prepared from human urine. Nearly complete 1H-NMR assignments were obtained for the 77 amino acid residues and partial assignments for the N-glycan and the glycosylphosphatidylinositol (GPI) anchor. These results together confirm that the C-terminal residue of the mature protein is Asn 77 and that the urine-derived form retains the nonlipid part of the GPI anchor. The data further indicate that the GPI anchor and possibly the N-glycan are structurally inhomogeneous and suggest that the phospholipid present in the intact GPI anchor was removed by phosphatidylinositol-specific phospholipase-D. The folding topology of the protein was determined from NOE enhancements and slowly exchanging backbone amide protons and consists primarily of five extended strands (denoted beta 1-beta 5 in sequence order), arranged into separate two-stranded (beta 1 and beta 2) and three-stranded (beta 3-beta 5) antiparallel beta-sheets. The same folding topology is found in all of the snake venom neurotoxins whose structures have been determined. The region between the beta 4 and beta 5 strands has helical character, a feature that is not present in the neurotoxins but that is seen in the topologically similar wheat germ agglutinin.     

Structural studies of alpha-bungarotoxin. 1. Sequence-specific 1H NMR resonance assignments

We report the complete sequence-specific assignment of the backbone resonances and most of the side-chain resonances in the 1H NMR spectrum of alpha-bungarotoxin by two-dimensional NMR. Problems with resonance overlap were resolved with the assistance of the HRNOESY experiment described in an accompanying paper [Basus, V.J., & Scheek, R.M. (1988) Biochemistry (second paper of three in this issue)]. Significant differences exist between the solution structure described here and the crystal structure of alpha-bungarotoxin, on the basis of the proton to proton distances obtained by nuclear Overhauser enhancement spectroscopy (NOESY) and the corresponding distances from the X-ray crystal structure [Love, R.A., & Stroud, R.M. (1986) Protein Eng. 1, 37]. These differences include a larger beta-sheet in solution and a different orientation of the invariant tryptophan, Trp-28, making the solution structure more consistent with the crystal structure of the homologous neurotoxin alpha-cobratoxin. Four errors in the order of the amino acids in the primary sequence were indicated by the NMR data. These errors were confirmed by chemical means, as described in an accompanying paper [Kosen, P.A., Finer-Moore, J., McCarthy, M.P., & Basus, V.J. (1988) Biochemistry (third paper of three in this issue)].     

Molecular recognition of human eosinophil-derived neurotoxin (RNase 2) by placental ribonuclease inhibitor

Placental ribonuclease inhibitor (RI) binds diverse mammalian RNases with dissociation constants that are in the femtomolar range. Previous studies on the complexes of RI with RNase A and angiogenin revealed that RI utilises largely distinctive interactions to achieve high affinity for these two ligands. Here we report a 2.0 angstroms resolution crystal structure of RI in complex with a third ligand, eosinophil-derived neurotoxin (EDN), and a mutational analysis based on this structure. The RI-EDN interface is more extensive than those of the other two complexes and contains a considerably larger set of interactions. Few of the contacts present in the RI-angiogenin complex are replicated; the correspondence to the RI-RNase A complex is somewhat greater, but still modest. The energetic contributions of various interface regions differ strikingly from those in the earlier complexes. These findings provide insight into the structural basis for the unusual combination of high avidity and relaxed stringency that RI displays.     

Glycan flexibility: insights into nanosecond dynamics from a microsecond molecular dynamics simulation explaining an unusual nuclear Overhauser effect

An atomistic all-atom molecular dynamics simulation of the trisaccharide β-d-ManpNAc-(1→4)[α-d-Glcp-(1→3)]-α-l-Rhap-OMe with explicit solvent molecules has been carried out. The trisaccharide represents a model for the branching region of the O-chain polysaccharide of a strain from Aeromonas salmonicida. The extensive MD simulations having a 1-μs duration revealed a conformational dynamics process on the nanosecond time scale, that is, a ‘time window’ not extensively investigated for carbohydrates to date. The results obtained from the MD simulation underscore the predictive power of molecular simulations in studies of biomolecular systems and also explain an unusual nuclear Overhauser effect originating from conformational exchange.     

Sequence of human eosinophil-derived neurotoxin cDNA: identity of deduced amino acid sequence with human nonsecretory ribonucleases

Several clones of human eosinophil-derived neurotoxin (EDN) cDNA have been isolated from a lambda gt10 cDNA library prepared from mRNA derived from noninduced HL-60 cells. The amino acid (aa) sequence deduced from the coding sequence of the EDN cDNA is identical to the aa sequence of urinary nonsecretory RNase. Comparison of the aa and/or nucleotide (nt) sequences of EDN and other proteins possessing ribonucleolytic activity, namely bovine seminal RNase, human and rat pancreatic RNases, eosinophil cationic protein (ECP), and human angiogenin, shows extensive identity at half-cystine residues and at aa of active sites. Differences in aa sequences at the active sites are often the result of single nt changes in the codons. The data presented here support the concept of a RNase gene superfamily containing secretory and nonsecretory RNases, angiogenin, EDN and ECP.     

Sequence-specific 1H-n.m.r. assignments and peptide backbone conformation in rat epidermal growth factor.

The solution conformation of rat epidermal growth factor (EGF) has been investigated by proton n.m.r. techniques. Two-dimensional proton n.m.r. experiments have allowed sequential resonance assignments to be made for most protons. On the basis of these assignments, two regions of anti-parallel beta-sheet structure have been derived from the n.m.r. data. A beta-sheet segment running from about V19 to V23 (capital letters refer to amino acids in the single-letter notation) is folded onto a beta-sheet segment running from R28 to N32 and joined by a chain reversal from E24 to D27. A second region involves a beta-turn from V34 to Y37, which starts a short beta-sheet up to G39, followed by a chain reversal up to Q43, which leads to folding of the C-terminal beta-sheet segment, i.e. H44-R45, running antiparallel to the short Y37 beta-sheet segment. The N-terminal segment up to G18 exists in a multiple bend conformation and is folded on to the V29-V23/R28-N32 beta-sheet such that Y10, Y13, Y22 and Y29 are proximal to each other. Structural comparison of rat, murine and human EGFs indicates a number of highly conserved structural features common to at least these species of EGF.     

Resonance assignments, solution structure, and backbone dynamics of the DNA- and RPA-binding domain of human repair factor XPA

XPA is involved in the damage recognition step of nucleotide excision repair (NER). XPA binds to other repair factors, and acts as a key element in NER complex formation. The central domain of human repair factor XPA (residues Met98 to Phe219) is responsible for the preferential binding to damaged DNA and to replication protein A (RPA). The domain consists of a zinc-containing subdomain with a compact globular structure and a C-terminal subdomain with a positively charged cleft in a novel alpha/beta structure. The resonance assignments and backbone dynamics of the central domain of human XPA were studied by multidimensional heteronuclear NMR methods. 15N relaxation data were obtained at two static magnetic fields, and analyzed by means of the model-free formalism under the assumption of isotropic or anisotropic rotational diffusion. In addition, exchange contributions were estimated by analysis of the spectral density function at zero frequency. The results show that the domain exhibits a rotational diffusion anisotropy (Dparallel/Dperpendicular) of 1.38, and that most of the flexible regions exist on the DNA binding surface in the cleft in the C-terminal subdomain. This flexibility may be involved in the interactions of XPA with various kinds of damaged DNA.     

An insertion in loop L7 of human eosinophil-derived neurotoxin is crucial for its antiviral activity

The human eosinophil granule ribonuclease, eosinophil‐derived neurotoxin (EDN) has been shown to have antiviral activity against respiratory syncytial virus‐B (RSV‐B). Other closely related and more active RNases such as RNase A, onconase, and RNase k6 do not have any antiviral activity. A remarkable unique feature of EDN is a nine‐residue insertion in its carboxy‐terminal loop, L7 which is not present in RNase A, and differs in sequence from the corresponding loop in another eosinophil RNase, eosinophil cationic protein (ECP). ECP has a much lower antiviral activity as compared to EDN. The current study probed the role of loop L7 of EDN in its antiviral activity. Three residues in loop L7, Arg117, Pro120, and Gln122, which diverge between EDN, ECP, and RNase A, were mutated to alanine alone and in combination to generate single, double, and triple mutants. These mutants, despite having RNase activity had decreased antiviral activity towards RSV suggesting the involvement of loop L7 in the interaction of EDN with RSV. It appears that the mutations in loop L7 disrupt the interaction of protein with the viral capsid, thereby inhibiting its entry into the virions. The study demonstrates that besides the RNase activity, loop L7 is another important determinant for the antiviral activity of EDN. J. Cell. Biochem. 113: 3104–3112, 2012. © 2012 Wiley Periodicals, Inc.