Edvards Liepinsh

NMR structure of the death domain of the p75 neurotrophin receptor

The intracellular domain of the p75 neurotrophin receptor (p75ICD) lacks catalytic activity but contains a motif similar to death domains found in the cytoplasmic regions of members of the tumor necrosis factor receptor family and their downstream targets. Although some aspects of the signaling pathways downstream of p75 have been elucidated recently, mechanisms of receptor activation and proximal signaling events are unknown. Here we report the nuclear magnetic resonance (NMR) structure of the 145 residue long p75ICD. The death domain of p75ICD consists of two perpendicular sets of three helices packed into a globular structure. The polypeptide segment connecting the transmembrane and death domains as well as the serine/threonine‐rich C‐terminal end are highly flexible in p75ICD. Unlike the death domains involved in signaling by the TNF receptor and Fas, p75ICD does not self‐associate in solution. A surface area devoid of charged residues in the p75ICD death domain may indicate a potential site of interaction with downstream targets.

Protein hydration studied with homonuclear 3D1H NMR experiments

SummaryHomonuclear 3D1H NOESY-TOCSY and 3D1H ROESY-TOCSY experiments were used to resolve and assign nuclear Overhauser effect (NOE) cross peaks between the water signal and individual polypeptide proton resonances in H2O solutions of the basic pancreatic trypsin inhibitor. Combined with a novel, robust water-suppression technique, positive and negative intermolecular NOEs were detected at 4°C. The observation of positive NOEs between water protons and protein protons enables more precise estimates of the very short residence times of the water molecules in the hydration sites on the protein surface.

NMR identification of hydrophobic cavities with ow water occupancies in protein structures using small gas molecules

Magnetization transfer through dipole-dipole interactions (nuclear Overhauser effects, NOEs) between water protons and the protons lining two small hydrophobic cavities in hen egg-white lysozyme demonstrates the presence of water molecules with occupancies of ∼10–50%. Similarly, NOEs were observed between the cavity protons and the protons of hydrogen, methane, ethylene or cyclopropane applied at 1–200 bar pressure. These gases can thus be used as general NMR indicators of empty or partially hydrated hydrophobic cavities in proteins. All gases reside in the cavities for longer than 1 ns in marked contrast to common belief that gas diffusion in proteins is not much slower than in water. Binding to otherwise empty cavities may be a major aspect of the anesthetic effect of small organic gas molecules.

NMR spectroscopy of hydroxyl protons in aqueous solutions of peptides and proteins

SummaryHydroxyl groups of serine and threonine, and to some extent also tyrosine are usually located on or near the surface of proteins. NMR observations of the hydroxyl protons is therefore of interest to support investigations of the protein surface in solution, and knowledge of the hydroxyl NMR lines is indispensable as a reference for studies of protein hydration in solution. In this paper, solvent suppression schemes recently developed for observation of hydration water resonances were used to observe hydroxyl protons of serine, threonine and tyrosine in aqueous solutions of small model peptides and the protein basic pancreatic trypsin inhibitor (BPTI). The chemical shifts of the hydroxyl protons of serine and threonine were found to be between 5.4 and 6.2 ppm, with random-coil shifts at 4°C of 5.92 ppm and 5.88 ppm, respectively, and those of tyrosine between 9.6 and 10.1 ppm, with a random-coil shift of 9.78 ppm. Since these spectral regions are virtually free of other polypeptide1H NMR signals, cross peaks with the hydroxyl protons are usually well separated even in homonuclear two-dimensional1H NMR spectra. To illustrate the practical use of hydroxyl proton NMR in polypeptides, the conformations of the side-chain hydroxyl groups in BPTI were characterized by measurements of nuclear Overhauser effects and scalar coupling constants involving the hydroxyl protons. In addition, hydroxyl proton exchange rates were measured as a function of pH, where simple first-order rate processes were observed for both acid- and base-catalysed exchange of all but one of the hydroxyl-bearing residues in BPTI. For the conformations of the individual Ser, Thr and Tyr side chains characterized in the solution structure with the use of hydroxyl proton NMR, both exact coincidence and significant differences relative to the corresponding BPTI crystal structure data were observed.[/p]

Protein hydration in aqueous solution

Proton nuclear magnetic resonance was used to study individual molecules of hydration water bound to the protein basic pancreatic trypsin inhibitor (BPTI) and to the nonapeptide oxytocin in aqueous solution. The experimental observations are nuclear Overhauser effects (NOE) between protons of individual amino acid residues of the protein and those of hydration water. These NOEs were recorded by two-dimensional (2D) and three dimensional (3D) NOE spectroscopy (NOESY) in the laboratory frame, and by the corresponding experiments in the rotating frame (ROESY). The studies show that there are two qualitatively different types of hydration sites. Four water molecules in the interior of the BPTI molecule are in identical locations in the crystal structure and in solution. Their NOEs with the protein protons are characterized by large negative cross-relaxation rates sigma NOE, which indicates that the residence times of the water molecules in these hydration sites are longer than ca. 10 ns. Additional experiments with extrinsic shift reagents established an upper limit of 20 ms at 4 degrees C for these residence times. Surface hydration of both the globular protein BPTI and the flexibly disordered polypeptide oxytocin is by water molecules with residence times in the subnanosecond range, as evidenced by small positive sigma NOE values observed for their NOEs with nearby polypeptide protons. Short residence times prevail for all surface hydration sites, independent of whether or not they are occupied by well ordered, X-ray observable water in the protein single crystals.

Inhibition of carnitine acetyltransferase by mildronate, a regulator of energy metabolism.

Carnitine acetyltransferase (CrAT; EC 2.3.1.7) catalyzes the reversible transfer of acetyl groups between acetyl-coenzyme A (acetyl-CoA) and L-carnitine; it also regulates the cellular pool of CoA and the availability of activated acetyl groups. In this study, biochemical measurements, saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy, and molecular docking were applied to give insights into the CrAT binding of a synthetic inhibitor, the cardioprotective drug mildronate (3-(2,2,2-trimethylhydrazinium)-propionate). The obtained results show that mildronate inhibits CrAT in a competitive manner through binding to the carnitine binding site, not the acetyl-CoA binding site. The bound conformation of mildronate closely resembles that of carnitine except for the orientation of the trimethylammonium group, which in the mildronate molecule is exposed to the solvent. The dissociation constant of the mildronate CrAT complex is approximately 0.1 mM, and the Ki is 1.6 mM. The results suggest that the cardioprotective effect of mildronate might be partially mediated by CrAT inhibition and concomitant regulation of cellular energy metabolism pathways.

Intramolecular C-H···O Hydrogen Bonding in 1,4-Dihydropyridine Derivatives

The diastereotopy of the methylene protons at positions 2 and 6 in 1,4-dihydropiridine derivatives with various substituents has been investigated. NMR spectroscopy and quantum chemistry calculations show that the CH···O intramolecular hydrogen bond is one of the factors amplifying the chemical shift differences in the 1H-NMR spectra.

A novel zinc-binding fold in the helicase interaction domain of the Bacillus subtilis DnaI helicase loader

The helicase loader protein DnaI (the Bacillus subtilis homologue of Escherichia coli DnaC) is required to load the hexameric helicase DnaC (the B. subtilis homologue of E. coli DnaB) onto DNA at the start of replication. While the C-terminal domain of DnaI belongs to the structurally well-characterized AAA+ family of ATPases, the structure of the N-terminal domain, DnaI-N, has no homology to a known structure. Three-dimensional structure determination by nuclear magnetic resonance (NMR) spectroscopy shows that DnaI presents a novel fold containing a structurally important zinc ion. Surface plasmon resonance experiments indicate that DnaI-N is largely responsible for binding of DnaI to the hexameric helicase from B. stearothermophilus, which is a close homologue of the corresponding much less stable B. subtilis helicase.

Contributions from hydration of carboxylate groups to the spectrum of water-polypeptide proton-proton Overhauser effects in aqueous solution

SummaryNuclear Overhauser effects (NOE) were measured between water protons and protons of the glutamic acid side chain of the bicyclic decapeptide