Structural insights into phenylethanolamines high-affinity binding site in NR2B from binding and molecular modeling studies

<p>Abstract</p> <p>Background</p> <p>Phenylethanolamines selectively bind to NR2B subunit-containing <it>N</it>-methyl-<it>D</it>-aspartate-subtype of ionotropic glutamate receptors and negatively modulate receptor activity. To investigate the structural and functional properties of the ifenprodil binding domain on the NR2B protein, we have purified a soluble recombinant rat NR2B protein fragment comprising the first ~400 amino acid amino-terminal domain (ATD2B) expressed in <it>E. coli</it>. Spectral measurements on refolded ATD2B protein demonstrated specific binding to ifenprodil. We have used site-directed mutagenesis, circular dichroism spectroscopy and molecular modeling to obtain structural information on the interactions between critical amino acid residues and ifenprodil of our soluble refolded ATD2B proteins. Ligand-induced changes in protein structure were inferred from changes in the circular dichroism spectrum, and the concentration dependence of these changes was used to determine binding constants for ifenprodil and its analogues.</p> <p>Results</p> <p>Ligand binding of ifenprodil, RO25,6981 and haloperidol on soluble recombinant ATD2B determined from circular dichroism spectroscopy yielded low-to-high micromolar equilibrium constants which concurred with functional IC₅₀measurement determined in heterologously expressed NR1/NR2B receptors in <it>Xenopus </it>oocytes. Amino acid residue substitutions of Asp101, Ile150 and Phe176 with alanine residue within the ATD2B protein altered the recombinant protein dissociation constants for ifenprodil, mirroring the pattern of their functional phenotypes. Molecular modeling of ATD2B as a clam-shell-like structure places these critical residues near a putative ligand binding site.</p> <p>Conclusion</p> <p>We report for the first time biochemical measurements show that the functional measurements actually reflect binding to the ATD of NR2B subunit. Insights gained from this study help advance the theory that ifenprodil is a ligand for the ATD of NR2B subunit.</p

W51 IRS 2: A Massive Jet Emerging from a Molecular Cloud into an H II Region

We have mapped [Ne II] (12.8um) and [S IV] (10.5um) emission from W51 IRS 2 with TEXES on Gemini North, and we compare these data to VLA free-free observations and VLT near-infrared images. With 0.5" spatial and 4 km/s spectral resolution we are able to separate the ionized gas into several components: an extended H II region on the front surface of the molecular cloud, several embedded compact H II regions, and a streamer of high velocity gas. We interpret the high velocity streamer as a precessing or fan-like jet, which has emerged from the molecular cloud into an OB star cluster where it is being ionized.Comment: 3 pages, 4 figures, 2 movie

An HST Snapshot Survey of Proto-Planetary Nebulae Candidates: Two Types of Axisymmetric Reflection Nebulosities

We report the results from an optical imaging survey of proto-planetary nebula candidates using the HST. We exploited the high resolving power and wide dynamic range of HST and detected nebulosities in 21 of 27 sources. All detected reflection nebulosities show elongation, and the nebula morphology bifurcates depending on the degree of the central star obscuration. The Star-Obvious Low-level-Elongated (SOLE) nebulae show a bright central star embedded in a faint, extended nebulosity, whereas the DUst-Prominent Longitudinally-EXtended (DUPLEX) nebulae have remarkable bipolar structure with a completely or partially obscured central star. The intrinsic axisymmetry of these proto-planetary nebula reflection nebulosities demonstrates that the axisymmetry frequently found in planetary nebulae predates the proto-planetary nebula phase, confirming previous independent results. We suggest that axisymmetry in proto-planetary nebulae is created by an equatorially enhanced superwind at the end of the asymptotic giant branch phase. We discuss that the apparent morphological dichotomy is caused by a difference in the optical thickness of the circumstellar dust/gas shell with a differing equator-to-pole density contrast. Moreover, we show that SOLE and DUPLEX nebulae are physically distinct types of proto-planetary nebulae, with a suggestion that higher mass progenitor AGB stars are more likely to become DUPLEX proto-planetary nebulae.Comment: 27 pages (w/ aaspp4.sty), 6 e/ps figures, 4 tables (w/ apjpt4.sty). Data images are available via ADIL (http://imagelib.ncsa.uiuc.edu/document/99.TU.01) To be published in Ap

Title Page Structural features of the glutamate binding site in recombinant NR1/NR2A N-methyl- D-aspartate receptors determined by site-directed mutagenesis and molecular modeling

Abbreviations: NMDA, N-methyl-D-aspartate; AMPA, α -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; TEVC, two electrode voltage clamp; D-AP5, D-2-amino-5-phosphonopentanoic acid; MTSEA, methanethiosulfonate ethylammonium. MOL 8185 3 Abstract We have used site-directed mutagenesis of amino acids located within the S1 and S2 ligand binding domains of the NR2A NMDA receptor subunit to explore the nature of ligand binding. Wild-type or mutated NR1/NR2A NMDA receptors were expressed in Xenopus laevis oocytes and studied using TEVC. We investigated the effects of mutations in the S1 and S2 regions on the potencies of the agonists L-glutamate, L-aspartate, RS-tetrazol-5-yl glycine and NMDA. Mutation of each of the corresponding residues found in the NR2A receptor subunit, suggested to be contact residues in the GluR2 AMPA receptor subunit, caused a rightward shift in the concentration-response curve for each agonist examined. None of the mutations examined altered the efficacy of glutamate as assessed by MTSEA potentiation of agonist-evoked currents. In addition none of the mutations altered the potency of glycine. Homology modeling and molecular dynamics were used to evaluate molecular details of ligand binding of both wild-type and mutant receptors, as well as to explore potential explanations for agonist selectivity between glutamate receptor subtypes. Our modeling studies support our interpretation of the mutagenesis data and indicate a similar binding strategy for L-glutamate and NMDA when they occupy the binding site in NMDA receptors as has been proposed for glutamate binding to the GluR2 AMPA receptor subunit. Furthermore, we offer an explanation as to why &apos;charge conserving&apos; mutations of two residues in the binding pocket result in non-functional receptor-channels and suggest a contributing molecular determinant of why NMDA is not an agonist at AMPA receptors. MOL 8185 4 The NMDA receptor-channel is thought to be formed from the co-assembly of two NR1 subunits and two NR2 subunits in a dimer of dimers configuration (Schorge and Colquhoun, 2003). NMDA receptors are unique among ligand-gated ion channels in that the binding of two different ligands is required for the activation of the receptor-channel complex. Glycine, a co-agonist, binds to residues located in the NR1 subunits, whereas glutamate binds to residues located in NR2 subunits (reviewed by X-ray diffraction studies have described the glycine binding domain of the NR1 subunit In this study we have systematically mutated all six of the predicted contact residues in either the S1 or S2 domains together with the histidine residue thought to play the same sitecapping role as Tyr450 in GluR2 Materials and Methods Plasmid constructs, cRNA synthesis and receptor expression in oocytes. The wild-type pSP64T-derived expression plasmids for NR1 and NR2A NMDA receptor subunits were as described in Oocytes were obtained from Xenopus laevis, after administration of a lethal dose of anaesthetic (all procedures were carried out in accordance with current UK Home Office requirements). Prior to injection with cRNAs of interest, the follicular membranes of the oocytes were removed. After injection oocytes were placed in separate wells of 24-well plates hours to allow for receptor expression and then stored at 4˚C until they were used for electrophysiological measurements. Electrophysiological Recordings and Solutions. TEVC recordings were made, using a GeneClamp 500 amplifier (Axon Instruments, Union City, CA), from oocytes that were placed in a modified frog Ringer solution that contained (in mM): NaCl 115, KCl 2.5, HEPES 10, BaCl 2 1.8; pH 7.3 with NaOH (20 ºC) (Sigma-Aldrich, UK). Current and voltage electrodes were made from thin-walled borosilicate glass (GC150TF-7.5, Harvard Apparatus, Kent, UK) using a PP-830 electrode puller (Narashige Instruments, Japan) and when filled with 3 M KCl possessed resistances of between 0.5 and 1.5 MΩ. Oocytes were voltageclamped at potentials of -40 mV. For L-glutamate, L-aspartate, NMDA and RS-tetrazol-5-yl glycine concentration-response measurements, the recording solution was further supplemented with 20 µM glycine and for glycine dose-response measurements this solution was supplemented with either 30 µM glutamate for wild-type receptors or 10 mM glutamate for mutant receptors. Glutamate receptor agonists were purchased from Tocris Cookson (Avonmouth, UK). Application of solutions was controlled manually and data were filtered at 10 Hz and digitized at 100 Hz. Test solutions were applied for 20 s or until a plateau to the agonist-evoked response had been achieved. The maximum agonist concentration applied to any oocyte was 30 mM as concentrations higher than this produced considerable changes in the osmolarity of the recording solution. The current response to a maximal agonist concentration for all constructs used here was always less than 3 µA at a holding potential of -40 mV. The open probability of NMDA receptors containing mutations in either the S1 or MOL 8185 8 S2 binding site was assessed by co-expressing these subunits with NR1 subunits carrying the A652C mutation where n H is the Hill coefficient, I max is the maximum current, [A] is the concentration of agonist, and EC 50 is the concentration of agonist that produces a half-maximum response. Each data point was then normalized to the fitted maximum of the dose-response curve. The normalized values were then pooled and averaged for each construct and fitted again with the Hill equation, with the maximum and minimum for each curve being constrained to asymptote to 1 and 0 respectively. A similar protocol was used to determine the concentration of the NMDA antagonist, D-AP5 required to inhibit a glutamate-evoked response by 50 % (IC 50 ). In these experiments the glutamate concentration was fixed at the concentration required to evoke a half-maximal response in the construct being investigated. Modeling of the glutamate binding site in the NR2A NMDA receptor subunit. A homology model of the S1-S2 region of NR2A (GenBank Accession D13211) was constructed from the NR1-glycine crystal structure (1PB7; In order to generate a binding site without close contacts and with reasonable hydrogen bonds, the homology model was supplemented with MMFF94 force field charges and subjected to Sybyl (version 6.9) molecular dynamics (MD) for 5000 femtoseconds (fs) using an NTP ensemble (20 K and 1 atm). During the first stage of molecular dynamics only the backbone atoms of the protein were permitted to move. A second molecular dynamics run with identical parameters allowed only the side chain atoms to move. Finally, the agonist ligand and all amino acid residues within 6.0 Å of it were subjected to the same treatment. In this way, all poor contacts within the protein were removed, while the ligand adopted reasonable hydrogen-bonding interactions with the surrounding residues. This allowed the model to achieve a relatively strain-free conformation. Subsequently, separate protein-ligand models of L-glutamate and N-methyl-D-aspartate (NMDA) were created with ligand conformations that closely matched that of L-glutamate from the GluR2 site (protein data bank 1FTJ). These were loaded with MMFF94 charges and manually docked into the candidate agonist binding site using the match command and the GluR2 site as guidance. The individual ligand-protein systems were subjected to molecular dynamics simulation in Sybyl (version 6.9) using the TRIPOS force field (20 K, 1 atm, NTP ensemble, coupling constant 20), for MOL 8185 10 10,000 fs holding fixed all residues outside of a 6.0 angstrom radius around the ligand aggregate. To produce the final structures, the last 1000 fs of molecular dynamics was averaged and subsequently minimized with the Powell algorithm until an energy gradient of 0.01 kcal/mol was reached. The resulting structures were used to evaluate changes in binding site interactions caused by mutation of binding site residues. For the mutation studies of R499K and D712E, modified structures were created using Sybyl&apos;s &quot;modify/mutate&quot; command on the optimized protein-ligand complexes described above. The mutated and liganded protein complexes were then subjected to the same molecular dynamics and energy minimization procedures employed for the native protein. All models can be downloaded from www.pharm.emory/straynelis/downloads. Further analysis of both NMDA and L-glutamate in the GluR2 structure and the NR2A model was performed using the GROMACS molecular dynamics package Results Steady MOL 8185 11 with values obtained from various studies of native and recombinant NMDA receptors (for a review see Dingledine et al., 1999). In addition, the mean maximal currents evoked by each of the agonists were similar, these being 1.77 ± 0.34 µA (tetrazol-5yl-glycine), 1.42 ± 0.23 µA (glutamate), 1.64 ± 0.20 µA (aspartate) and 1.66 ± 0.17 µA (NMDA). Evaluation of responses to maximally effective concentrations suggests that agonist efficacy relative to glutamate for all compounds ranged between 89 -97 % (data not shown). The ligand binding site in the NR2A receptor subunit. We created a homology model of the NR2A S1-S2 ligand binding domain using the NR1-glycine crystal structure as a template Glutamate and its analogues were docked into the NR2A model using the original description of glutamate binding to AMPA receptor S1-S2 (Armstrong and Gouaux, 2000) as a guide and molecular dynamics as a provisional refinement protocol (see Materials and Methods). We subsequently docked NMDA into our model of NR2A and found several notable differences in binding. The Tyr711 orientation is assisted by H-bonding to Glu394. Given that in GluR2 Met708 NMDA receptor subunits is conserved in the GluR2 AMPA receptor subunit. It has been reported previously NMDA receptor subunit to glycine (S664G) reduces glutamate potency by 180-fold. Thus, we first made the equivalent serine to glycine mutation in the NR2A NMDA receptor subunit. Previous studies have characterized the properties of glutamate-activated NR1/NR2A(T671A) receptors and the homologous mutation in NR2D-containing NMDA receptors at both the whole-cell and single-channel level We interpret the effect on potency of the glycine, but not alanine, substitution at NR2A Ser670 as a likely indication of alterations in the geometry of the backbone chain. Glycine, which increases chain flexibility, may allow the chain to adopt conformations that hinder the ability of a hydrogen bond to form between the Ser670 chain nitrogen and the α- carboxyl. This interaction may help stabilize the other interactions of Ser670 and Thr671 with the γ-carboxyl (see MOL 8185 17 between the potency of NMDA and glutamate for NR2A(T671A) containing receptors. Given a possible reduction in degrees of freedom and different orientations in the cleft, we predict that shorter D-aspartate based ligands may require less stabilization of their γ-carboxyl. Mutation of residues in the S1 binding domain. Three uncharged residues in the S1 binding domain (H466, S492 and T494) were separately replaced with alanine by mutagenesis. Each of these residues is conserved in the four NR2 NMDA receptor subunits, while in the corresponding GluR2 AMPA subunit a tyrosine residue replaces histidine and a proline is found at the homologous position occupied by serine. Thr494 is conserved in each of these subunits as well as in the NR1 subunit (see The biggest shift in potency for tetrazol-5yl-glycine was seen with the H466A mutation, T494A gave the biggest shifts in glutamate and aspartate potencies, while the biggest shift in NMDA potency was observed upon mutation of the Ser492 residue. In general these three mutations had less effect on tetrazol-5yl-glycine potency with only between 21-and 42-fold shifts in potency being observed when compared to wild-type receptors. We predict that tetrazol-5yl-glycine will show additional hydrogen bonds between the tetrazol ring and residues within the binding pocket (modeling data not shown), suggesting this agonist utilizes additional features of the binding pocket to stabilize its interactions. We interpret these additional interactions as stabilizing forces that counteract the loss of Ser492 and Thr494 interactions. NR2A(S492A S670G) has additive effects on glutamate potency. We also investigated the effects of introducing two mutations in different lobes into the binding site of MOL 8185 18 the NR2A NMDA receptor subunit. The two double mutations chosen were NR2A(S492A S670G) and NR2A(T494A T671A). As is shown in essentially abolished glutamate sensitivity, as only very small currents could be evoked by glutamate even when the concentration of agonist was raised to 30 mM. The ability of the double mutant to reduce potency further than each single mutant in isolation suggests that each of these mutations, individually, may not dramatically alter contacts of the other serine and threonine residues within the binding site. Mutations in S1 and S2 domains do not affect agonist efficacy. Changes in the potency (EC 50 ) of an agonist at receptors carrying mutations are difficult to ascribe to changes in agonist binding if the mutation also affects the agonist&apos;s efficacy i.e. its ability to gate the channel (see . For this reason we present several lines of evidence as to why we consider the main effects of the mutations we have described above are to alter the ability of glutamate (and other agonists) to bind to the NMDA receptor. First, to examine the ability of mutations in the S1 and S2 binding domains to affect the ability of glutamate to gate the channel once bound to the receptor we co-expressed wildtype and mutant NR2A subunits with NR1 subunits carrying the A652C mutation MOL 8185 19 residues located in the S1 Second and in contrast to their effects on the potency for glutamate analogues, none of the mutations studied here affected the potency of glycine Finally, for each of the mutations made in either the S1 or S2 domain we did not observe &gt; 5-fold changes in the maximal currents that were evoked in mutated NR2 subunits when compared to wild-type responses. The fact that large differences were not observed and also given that there were no significant decreases in the Hill Slopes (see Mutation of NR2A S1 residues to the homologous residues in NR1. Two of the proposed residues in the S1 domain, thought to participate in the binding of glutamate, vary between the NR2 and NR1 subunits; namely a serine at position 492 in NR2A (a proline residue in NR1 and GluR2) and a histidine at position 466 in NR2A (a phenylalanine residue in NR1). Our modeling studies suggest that the amino group of NMDA forms a hydrogen bond with the backbone oxygen of Ser492 His466 occupies a position analogous to Tyr450 in GluR2, and likely provides the binding pocket with a similar electron polarizable ring. MOL 8185 22 effects may arise as a result of ring twisting. The lower potency shift in the case of the H466F mutation may owe its origin to such a phenomenon. NR2A(R499K) and NR2A(D712E) produce non-functional receptor-channels. Two of the six hypothetical contact residues in the binding site are charged. We introduced charge-conserving mutations at position 499 (arginine to lysine; R499K) and position 712 (aspartate to glutamate; D712E). When co-injected with cRNA for the NR1 NMDA receptor subunit neither of these constructs gave currents when glutamate was applied up to 10 mM. We also investigated whether any of the other three ligands used in this study might activate either NR2A(R499K)-or NR2A(D712E)-containing NMDA receptor-channels. In all cases each of these ligands failed to evoke a response in oocytes injected with these two constructs. It has previously been reported that both the equivalent arginine to lysine mutation in the NR2B NMDA receptor subunit (R493K; Laube et al., 1997) and the equivalent aspartate to glutamate mutation in NR2A/NR2B NMDA receptor subunits (D731E and D732E; In a separate vein, the NR2A model provides a satisfying interpretation of the consequences of replacing Asp712 with Glu. In the wild-type receptor Asp712 makes three coupled hydrogen bonds, two of which are to the neutral OH groups in the side chains of Ser492 and Tyr742 (not shown in subunit to an Asn residue also abolishes receptor function. Thus it appears that conservative substitution (with Glu) or substitution with either small non-polar residues or polar residues each result in non-functional receptors. Hence these two non-functional mutations serve to illustrate important geometric constraints within the binding pocket. Discussion Effects of S1 and S2 mutations on agonist potency. Several structure-function studies of ionotropic glutamate receptors and related kainate binding proteins have identified residues located in the S1 and S2 binding domains that, when mutated, lead to a reduction in the ability of the ligand to remain bound to its binding site (for example see MOL 8185 24 While shifts in the EC 50 of a ligand for the activation of a receptor or a change in &apos;apparent affinity&apos; of a ligand measured in a binding assay cannot indicate, definitively, that the mutation has had a direct effect on the microscopic rate constants determining binding (see Further evidence that the S1 and S2 mutations studied have their major effect on agonist Taken together these results support our hypothesis that the shifts in EC 50 we report largely reflect changes in binding rates. Each of the point mutations investigated in this study caused a decrease in potency for all agonists examined. However the degrees of shift observed were, in general, greater for Even with knowledge that a residue is located in the binding pocket and is likely to hydrogen bond directly with glutamate, structure-function studies may miss, or place lesser emphasis on, a residue when only a small decrease in agonist potency is observed. This is exemplified when we consider the Ser670 residue in the S2 domain. In our present study we have shown that mutation of Ser670 to glycine, in the NR2A NMDA receptor subunit causes a far greater shift in potency than that seen when the residue is mutated to an alanine residue MOL 8185 26 Modeling of the binding site in the NR2A NMDA receptor subunit. A primary goal of this study is to provide functional data supporting the emerging idea that the glutamate binding site shares similar molecular features across the glutamate receptor family. The increasing availability of crystallographic data and homology modeling has increased the need for functional studies to substantiate and interpret data. Our experiments represent a systematic exploration of all proposed contact residues in a single NMDA subunit (NR2A). Furthermore, we studied four ligands at all mutant receptors, which provides additional insight into the nature of the binding pocket. Several clear themes emerge from these data. First, we find that the major molecular determinants of glutamate binding to GluR2 (see This is most notable when we mutated the two charged residues in the binding site. Shortening the side chain length of Arg499 by changing this to lysine increases the distance between its side chain and the α-carboxyl of glutamate to such an extent that it cannot hydrogen bond. Similarly increasing the amount to which the γ-carboxyl of the residue at position 712 protrudes into the binding site by swapping the aspartate residue for glutamate indicates that length of the side chain at this position is critical for a functional receptor. Third, we describe several hypothetical features of the NMDA receptor binding pocket that define its selectivity for aspartate derivatives compared to AMPA receptors. Finally we propose that the differential contribution of residues to stabilization of different ligands MOL 8185 27 explains the differing effects of mutants on the rank order of potency -for example the relatively small shifts in potency of tetrazol-5yl-glycine may result from its ability to hydrogen bond with (additional) groups in the binding site which counteract the loss of the sites utilized by the other ligands. Summary. Although these conclusions appear straightforward and predictive, we are aware of the limitations of homology modeling, particularly for the glutamate binding pocket which shows a number of interesting features we have neglected. Specifically, we have not included potential water bridged interactions in the pocket, and at times needed to choose between one of several possible orientations for side chains. Despite these caveats, the data presented here answer several longstanding questions about glutamate receptor pharmacology, and make a number of predictions that will help focus additional questions and experiments on the glutamate receptor-binding domain

Near-Infrared and Optical Observations of Type Ic SN 2021krf: Luminous Late-time Emission and Dust Formation

We present near-infrared (NIR) and optical observations of the Type Ic supernova (SN Ic) SN 2021krf obtained between days 13 and 259 at several ground-based telescopes. The NIR spectrum at day 68 exhibits a rising $K$-band continuum flux density longward of $\sim$ 2.0 $\mu$m, and a late-time optical spectrum at day 259 shows strong [O I] 6300 and 6364 \r{A} emission-line asymmetry, both indicating the presence of dust, likely formed in the SN ejecta. We estimate a carbon-grain dust mass of $\sim$ 2 $\times$ 10$^{-5}$ M$_{\odot}$ and a dust temperature of $\sim$ 900 - 1200 K associated with this rising continuum and suggest the dust has formed in SN ejecta. Utilizing the one-dimensional multigroup radiation hydrodynamics code STELLA, we present two degenerate progenitor solutions for SN 2021krf, characterized by C-O star masses of 3.93 and 5.74 M$_{\odot}$, but with the same best-fit $^{56}$Ni mass of 0.11 M$_{\odot}$ for early times (0-70 days). At late times (70-300 days), optical light curves of SN 2021krf decline substantially more slowly than that expected from $^{56}$Co radioactive decay. Lack of H and He lines in the late-time SN spectrum suggests the absence of significant interaction of the ejecta with the circumstellar medium. We reproduce the entire bolometric light curve with a combination of radioactive decay and an additional powering source in the form of a central engine of a millisecond pulsar with a magnetic field smaller than that of a typical magnetar.Comment: Accepted for publication in ApJ, 27 pages, 21 figures, 6 tables. Previous arXiv submission (arXiv:2211.00205) replaced after acceptanc

The phase diagram of NiSi under the conditions of small planetary interiors

The phase diagram of NiSi has been determined using in situ synchrotron X-ray powder diffraction multi-anvil experiments to 19 GPa, with further preliminary results in the laser-heated diamond cell reported to 60 GPa. The low-pressure MnP-structured phase transforms to two different high-pressure phases depending on the temperature: the ε-FeSi structure is stable at temperatures above ∼1100 K and a previously reported distorted-CuTi structure (with Pmmn symmetry) is stable at lower temperature. The invariant point is located at 12.8 ± 0.2 GPa and 1100 ± 20 K. At higher pressures, ε -FeSi-structured NiSi transforms to the CsCl structure with CsCl-NiSi as the liquidus phase above 30 GPa. The Clapeyron slope of this transition is -67 MPa/K. The phase boundary between the ε -FeSi and Pmmn structured phases is nearly pressure independent implying there will be a second sub-solidus invariant point between CsCl, ε -FeSi and Pmmn structures at higher pressure than attained in this study. In addition to these stable phases, the MnP structure was observed to spontaneously transform at room temperature to a new orthorhombic structure (also with Pnma symmetry) which had been detailed in previous ab initio simulations. This new phase of NiSi is shown here to be metastable