Linda K. Nicholson

Pulse sequences for removal of the effects of cross correlation between dipolar and chemical-shift anisotropy relaxation mechanisms on the measurement of heteronuclear T1 and T2 values in proteins

The effects of cross correlation between dipolar and chemical-shift anisotropy relaxation interactions on the measurement of heteroatom T1 and T2 relaxation times in proteins is considered. It is shown that such effects can produce errors of approximately 25% in the measurement of 15N transverse relaxation times at a field strength of 11.8 T. Cross correlation has a less significant effect on the measurement of 15N spin-lattice relaxation rates and for proteins the errors in T1 decrease as a function of increasing molecular weight. Nevertheless, for T1 measurements at 11.8 T errors of approximately 15 and 5% are calculated for proteins with correlation times, τc, of 5 and 9 ns, respectively. Pulse sequences which eliminate dipolar and chemical-shift anisotropy cross-correlation effects are described. These sequences are used to make more accurate measurements of 15N T1 and T2 values of staphylococcal nuclease and to determine errors in these parameters that result when cross correlations are present.

The prolyl isomerase Pin1 regulates amyloid precursor protein processing and amyloid-β production

Neuropathological hallmarks of Alzheimers disease are neurofibrillary tangles composed of tau and neuritic plaques comprising amyloid-β peptides (Aβ) derived from amyloid precursor protein (APP), but their exact relationship remains elusive. Phosphorylation of tau and APP on certain serine or threonine residues preceding proline affects tangle formation and Aβ production in vitro. Phosphorylated Ser/Thr-Pro motifs in peptides can exist in cis or trans conformations, the conversion of which is catalysed by the Pin1 prolyl isomerase. Pin1 has been proposed to regulate protein function by accelerating conformational changes, but such activity has never been visualized and the biological and pathological significance of Pin1 substrate conformations is unknown. Notably, Pin1 is downregulated and/or inhibited by oxidation in Alzheimers disease neurons, Pin1 knockout causes tauopathy and neurodegeneration, and Pin1 promoter polymorphisms appear to associate with reduced Pin1 levels and increased risk for late-onset Alzheimers disease. However, the role of Pin1 in APP processing and Aβ production is unknown. Here we show that Pin1 has profound effects on APP processing and Aβ production. We find that Pin1 binds to the phosphorylated Thr 668-Pro motif in APP and accelerates its isomerization by over 1,000-fold, regulating the APP intracellular domain between two conformations, as visualized by NMR. Whereas Pin1 overexpression reduces Aβ secretion from cell cultures, knockout of Pin1 increases its secretion. Pin1 knockout alone or in combination with overexpression of mutant APP in mice increases amyloidogenic APP processing and selectively elevates insoluble Aβ42 (a major toxic species) in brains in an age-dependent manner, with Aβ42 being prominently localized to multivesicular bodies of neurons, as shown in Alzheimers disease before plaque pathology. Thus, Pin1-catalysed prolyl isomerization is a novel mechanism to regulate APP processing and Aβ production, and its deregulation may link both tangle and plaque pathologies. These findings provide new insight into the pathogenesis and treatment of Alzheimers disease.

Flexibility and function in HIV-1 protease

HIV protease is a homodimeric protein whose activity is essential to viral function. We have investigated the molecular dynamics of the HIV protease, thought to be important for proteinase function, bound to high affinity inhibitors using NMR techniques. Analysis of 15N spin relaxation parameters, of all but 13 backbone amide sites, reveals the presence of significant internal motions of the protein backbone. In particular, the flaps that cover the proteins active site of the protein have terminal loops that undergo large amplitude motions on the ps to ns time scale, while the tips of the flaps undergo a conformational exchange on the μs time scale. This enforces the idea that the flaps of the proteinase are flexible structures that facilitate function by permitting substrate access to and product release from the active site of the enzyme.

RANTES Binding and Down-Regulation by a Novel Human Herpesvirus-6 β Chemokine Receptor

The human herpesvirus 6 (HHV-6) U51 gene defines a new family of betaherpesvirus-specific genes encoding multiple transmembrane glycoproteins with similarity to G protein-coupled receptors, in particular, human chemokine receptors. These are distinct from the HHV-6 U12 and HCMV US28 family. In vitro transcription and translation as well as transient cellular expression of U51 showed properties of a multiple transmembrane protein with a 30-kDa monomer as well as high m.w. aggregates or oligomers. Transient cellularly expressed U51 also appeared to form dimeric intermediates. Despite having only limited sequence similarity to chemokine receptors, U51 stably expressed in cell lines showed specific binding of the CC chemokine RANTES and competitive binding with other β chemokines, such as eotaxin; monocyte chemoattractant protein 1, 3, and 4; as well as the HHV-8 chemokine vMIPII. In epithelial cells already secreting RANTES, U51 expression resulted in specific transcriptional down-regulation. This correlated with reduced secretion of RANTES protein into the culture supernatants. Regulation of RANTES levels may alter selective recruitment of circulating inflammatory cells that the virus can infect and thus could mediate the systemic spread of the virus from initial sites of infection in epithelia. Alternatively, chemokine regulation could modulate a protective inflammatory response to aid the spread of virus by immune evasion. Such mimicry, by viral proteins, of host receptors leading to down-regulation of chemokine expression is a novel immunomodulatory mechanism.

Repeated domains of leptospira immunoglobulin-like proteins interact with elastin and tropoelastin.

Leptospira spp., the causative agents of leptospirosis, adhere to components of the extracellular matrix, a pivotal role for colonization of host tissues during infection. Previously, we and others have shown that Leptospira immunoglobulin-like proteins (Lig) of Leptospira spp. bind to fibronectin, laminin, collagen, and fibrinogen. In this study, we report that Leptospira can be immobilized by human tropoelastin (HTE) or elastin from different tissues, including lung, skin, and blood vessels, and that Lig proteins can bind to HTE or elastin. Moreover, both elastin and HTE bind to the same LigB immunoglobulin-like domains, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12 as demonstrated by enzyme-linked immunosorbent assay (ELISA) and competition ELISAs. The LigB immunoglobulin-like domain binds to the 17th to 27th exons of HTE (17–27HTE) as determined by ELISA (LigBCon4, KD = 0.50 μm; LigBCen7′–8, KD = 0.82 μm; LigBCen9, KD = 1.54 μm; and LigBCen12, KD = 0.73 μm). The interaction of LigBCon4 and 17–27HTE was further confirmed by steady state fluorescence spectroscopy (KD = 0.49 μm) and ITC (KD = 0.54 μm). Furthermore, the binding was enthalpy-driven and affected by environmental pH, indicating it is a charge-charge interaction. The binding affinity of LigBCon4D341N to 17–27HTE was 4.6-fold less than that of wild type LigBCon4. In summary, we show that Lig proteins of Leptospira spp. interact with elastin and HTE, and we conclude this interaction may contribute to Leptospira adhesion to host tissues during infection.

Solution structure of ThiS and implications for the evolutionary roots of ubiquitin

ThiS is a sulfur carrier protein that plays a central role in thiamin biosynthesis in Escherichia coli. Here we report the solution NMR structure of ThiS, the first for this class of sulfur carrier proteins. Although ThiS shares only 14% sequence identity with ubiquitin, it possesses the ubiquitin fold. This structural homology, combined with established functional similarities involving sulfur chemistry, demonstrates that the eukaryotic ubiquitin and the prokaryotic ThiS evolved from a common ancestor. This illustrates how structure determination is essential in establishing evolutionary links between proteins in which structure and function have been conserved through eons of evolution despite loss of sequence identity. The ThiS structure reveals both hydrophobic and electrostatic surface features that are likely determinants for interactions with binding partners. Comparison with surface features of ubiquitin and ubiquitin homologs SUMO-1, RUB-1 and NEDD8 suggest how Nature has utilized this single fold to incorporate similar chemistry into a broad array of highly specific biological processes.

Essential role for the prolyl isomerase Pin1 in Toll-like receptor signaling and type I interferon-mediated immunity

Toll-like receptors (TLRs) shape innate and adaptive immunity to microorganisms. The enzyme IRAK1 transduces signals from TLRs, but mechanisms for its activation and regulation remain unknown. We found here that TLR7 and TLR9 activated the isomerase Pin1, which then bound to IRAK1; this resulted in activation of IRAK1 and facilitated its release from the receptor complex to activate the transcription factor IRF7 and induce type I interferons. Consequently, Pin1-deficient cells and mice failed to mount TLR-mediated, interferon-dependent innate and adaptive immune responses. Given the critical role of aberrant activation of IRAK1 and type I interferons in various immune diseases, controlling IRAK1 activation via inhibition of Pin1 may represent a useful therapeutic approach.

An improved method for distinguishing between anisotropic tumbling and chemical exchange in analysis of 15N relaxation parameters

Although an accurate description of global tumbling of a protein is essential for correct analysis of internal motions, proper distinction between the effects of anisotropic rotational diffusion and conformational exchange has remained a challenge. We present a novel two-part filtering procedure designed specifically to distinguish between the effects of anisotropy and conformational exchange. The efficacy of this method is assessed using synthetic data sets. The method is then applied to two proteins of dramatically different size and shape, OspA and SH3. The large size and extreme anisotropy of OspA provide a challenging case, where conformational exchange is a small perturbation of the effects of anisotropy on transverse relaxation rates. Conversely, in the chicken c-Src SH3 domain, with its small size and nearly spherical shape, anisotropy is a small perturbation of the effects of conformational exchange on transverse relaxation rates. Accurate extraction of the global tumbling parameters for each protein allows optimal characterization of conformational exchange processes, as well as ps–ns time scale motions.

Fibronectin binds to and induces conformational change in a disordered region of leptospiral immunoglobulin-like protein B

Leptospira interrogans is a pathogenic spirochete that causes disease in both humans and animals. LigB (Leptospiral immunoglobulin-like protein B) contributes to the binding of Leptospira to extracellular matrix proteins such as fibronectin (Fn), fibrinogen, laminin, and collagen. A high affinity Fn-binding region of LigB has been recently localized to LigBCen2, which contains the partial eleventh and full twelfth immunoglobulin-like repeats (LigBCen2R) and 47 amino acids of the non-repeat region (LigBCen2NR) of LigB. In this study, LigBCen2NR was shown to bind to the N-terminal domain (NTD) of Fn (KD = 379 nm) by an enzyme-linked immunosorbent assay and isothermal titration calorimetry. Interestingly, this sequence was not observed to adopt secondary structure by far UV circular dichroism or by differential scanning calorimetry, in agreement with computer-based secondary structure predictions. A low partition coefficient (Kav) measured with gel permeation chromatography, a high hydrodynamic radius (Rh) measured with dynamic light scattering, and the insensitivity of the intrinsic viscosity to guanidine hydrochloride treatment all suggest that LigBCen2NR possesses an extended and disordered structure. Two-dimensional 15N-1H HSQC NMR spectra of intact LigBCen2 in the absence and presence of NTD are consistent with these observations, suggesting the presence of both a β-rich region and an unstructured region in LigBCen2 and that the latter of these selectively interacts with NTD. Upon binding to NTD, LigBCen2NR was observed by CD to adopt a β-strand-rich structure, suggestive of the known β-zipper mode of NTD binding.

EXPERIMENTAL DETERMINATION OF TORSION ANGLES IN THE POLYPEPTIDE BACKBONE OF THE GRAMICIDIN A CHANNEL BY SOLID STATE NUCLEAR MAGNETIC RESONANCE

An analytical method for the determination of torsion angles from solid state 15N nuclear magnetic resonance (n.m.r.) spectroscopic data is demonstrated. Advantage is taken of the 15N-1H and 15N-13C dipolar interactions as well as the 15N chemical shift interaction in oriented samples. The membrane-bound channel conformation of gramicidin A has eluded an atomic resolution structure determination by more traditional approaches. Here, the torsion angles for the Ala3 site are determined by obtaining the n.m.r. data for both the Gly2-Ala3 and Ala3-Leu4 peptide linkages. Complete utilization of the orientational constraints derived from these orientation-dependent nuclear spin interactions in restricting the conformational space is most effectively achieved by utilizing spherical trigonometry. Two possible sets of torsion angles for the Ala3 site are obtained (phi, psi = -129 degrees, 153 degrees and -129 degrees, 122 degrees), both of which are consistent with a right-handed beta-helix. Other functional and computational evidence strongly supports the set for which the carbonyl oxygen atom of the Ala3-Leu4 linkage is rotated into the channel lumen.