Darón I. Freedberg

Flap opening and dimer-interface flexibility in the free and inhibitor-bound HIV protease, and their implications for function

BACKGROUNDn(1)H and (15)N transverse relaxation measurements on perdeuterated proteins are ideally suited for detecting backbone conformational fluctuations on the millisecond-microsecond timescale. The identification of conformational exchange on this timescale by measuring the relaxation of both (1)H and (15)N holds great promise for the elucidation of functionally relevant conformational changes in proteins.nnnRESULTSnWe measured the transverse (1)H and (15)N relaxation rates of backbone amides of HIV-1 protease in its free and inhibitor-bound forms. An analysis of these rates, obtained as a function of the effective rotating frame field, provided information about the timescale of structural fluctuations in several regions of the protein. The flaps that cover the active site of the inhibitor-bound protein undergo significant changes of backbone (φ,psi) angles, on the 100 micros timescale, in the free protein. In addition, the intermonomer beta-sheet interface of the bound form, which from protease structure studies appears to be rigid, was found to fluctuate on the millisecond timescale.nnnCONCLUSIONSnWe present a working model of the flap-opening mechanism in free HIV-1 protease which involves a transition from a semi-open to an open conformation that is facilitated by interaction of the Phe53 ring with the substrate. We also identify a surprising fluctuation of the beta-sheet intermonomer interface that suggests a structural requirement for maturation of the protease. Thus, slow conformational fluctuations identified by (1)H and (15)N transverse relaxation measurements can be related to the biological functions of proteins.

Extracellular structure of polysialic acid explored by on cell solution NMR

The capsular polysaccharide of the pathogens Neisseria meningitidis serogroup B and of Escherichia coli K1, α(2 → 8) polysialic acid (PSA), is unusual, because when injected into adult humans, it generates little or no antibody. In contrast, people infected with these pathogens generate specific serum antibodies. A structural study on cells is used to address this anomaly by characterizing antigen structures in vivo. We introduce on cell multidimensional solution NMR spectroscopy for direct observation of PSA on E. coli bacteria. Using 13C,15N-labeled PSA, we applied a combination of heteronuclear NMR methods, such as heteronuclear single quantum coherence, HNCA, and HNCO, in vivo. Analysis reveals that free and cell-bound PSA are structurally similar, indicating that the poor immunogenicity of PSA is not due to major structural differences between cells and purified PSA. The 13C linewidths of PSA on cells are 2 to 3 times larger than the corresponding ones in free PSA. The possible implications of the differences between free and on cell PSA are discussed. In addition, we demonstrate the suitability of the method for in vivo kinetic studies.

More accurate 1JCH coupling measurement in the presence of 3JHH strong coupling in natural abundance

J couplings are essential for measuring RDCs (residual dipolar couplings), now routinely used to deduce molecular structure and dynamics of glycans and proteins. Accurate measurement of (1)J(CH) is critical for RDCs to reflect the true structure and dynamics in the molecule of interest. We report noticeable discrepancies between (1)J(CH) values measured with HSQC type pulse sequences in the (1)H dimension from those measured in the (13)C dimension for 17 sugars and show that these discrepancies arise from strong scalar coupling. In order to determine how to minimize errors in measuring (1)J(CH), we analyze the strong coupling effects in detail using the product operator-formalism and spectral simulations based on the solution of the Liouville equation (not considering relaxation effects) in the presence of strong coupling. We report that the apparent (1)J(CH) measured with 2D HSQC-based sequences in either dimension can be in error by up to 4 Hz and that the values measured in the (1)H dimension can disagree with those in the (13)C dimension by up to 7 Hz. We demonstrate that spectral simulations can reproduce the errors induced by strong coupling and that these can be used to extract true (1)J(CH) values. We find that the (1)J(CH) values measured using a modified Z-filtered coupled HSQC are still affected by strong coupling. We conclude that spectral simulation yields accurate (1)J(CH) with errors as low as 1% in the presence of strong coupling.

Letter to the Editor: Sequential resonance assignments of the extracellular ligand binding domain of the human TGF-β type II receptor

Key words: ligand, NMR assignments, receptor, TGF, Transforming growth factor-beta, type II receptorBiological contextTransforming growth factor beta (TGF-b) is a potentgrowth suppressor of many different normal cell types(Massague, 1998). Significantly, many cancer celltypes show a diminished sensitivity to TGF-b medi-ated growthinhibition. In addition to its important rolein growth regulation, TGF-b also affects the adhesiveproperties of cells by regulating key components in-volvedincell adhesion.TGF-bsignalingis initiatedbybinding of growth factor ligand to two related single-passtransmembranereceptorserine/threoninekinases,known as the TGF-btype I (TbR1) and type II (TbR2)receptors. In the sequential model for TGF-b receptoractivation, growth factor ligand first binds to TbR2.TbR1, which is incapable of binding ligand in theabsence of TbR2, associates with the TGF-bx0fTbR2complex and is activated by the constitutive kinaseactivity of TbR2 (Massague, 1998).Presently, the three-dimensional structure of eachof the major TGF-b isoforms (TGF-b1, -b2, and-b3) is known (reviewed by Massague, 1998). Thereis no experimentally determined three-dimensionalstructural information available regarding the ligandbinding domains of the TGF-b receptors, although X-ray crystal structures of extracellular ligand bindingdomain of the activin type II receptor and the extracel-lular domain of the bone morphogenic protein (BMP)receptor 1A bound to the TGF-bsuperfamily memberBMP-2 have recently been reported (Greenwald et al.,1999; Kirsch et al., 2000). Herein, we report backbone

NMR of glycans: Shedding new light on old problems

The diversity in molecular arrangements and dynamics displayed by glycans renders traditional NMR strategies, employed for proteins and nucleic acids, insufficient. Because of the unique properties of glycans, structural studies often require the adoption of a different repertoire of tailor-made experiments and protocols. We present an account of recent developments in NMR techniques that will deepen our understanding of structure-function relations in glycans. We open with a survey and comparison of methods utilized to determine the structure of proteins, nucleic acids and carbohydrates. Next, we discuss the structural information obtained from traditional NMR techniques like chemical shifts, NOEs/ROEs, and coupling-constants, along with the limitations imposed by the unique intrinsic characteristics of glycan structure on these approaches: flexibility, range of conformers, signal overlap, and non-first-order scalar (strong) coupling. Novel experiments taking advantage of isotopic labeling are presented as an option for overcoming spectral overlap and raising sensitivity. Computational tools used to explore conformational averaging in conjunction with NMR parameters are described. In addition, recent developments in hydroxyl detection and hydrogen bond detection in protonated solvents, in contrast to traditional sample preparations in D2O for carbohydrates, further increase the tools available for both structure information and chemical shift assignments. We also include previously unpublished data in this context. Accurate determination of couplings in carbohydrates has been historically challenging due to the common presence of strong-couplings. We present new strategies proposed for dealing with their influence on NMR signals. We close with a discussion of residual dipolar couplings (RDCs) and the advantages of using (13)C isotope labeling that allows gathering one-bond (13)C-(13)C couplings with a recently improved constant-time COSY technique, in addition to the commonly measured (1)H-(13)C RDCs.

Constant time INEPT CT-HSQC (CTi-CT-HSQC) - A new NMR method to measure accurate one-bond J and RDCs with strong 1H-1H couplings in natural abundance.

Strong (1)H-(1)H coupling can significantly reduce the accuracy of (1)J(CH) measured from frequency differences in coupled HSQC spectra. Although accurate (1)J(CH) values can be extracted from spectral simulation, it would be more convenient if the same accurate (1)J(CH) values can be obtained experimentally. Furthermore, simulations reach their limit for residual dipolar coupling (RDC) measurement, as many significant, but immeasurable RDCs are introduced into the spin system when a molecule is weakly aligned, thus it is impossible to have a model spin system that truly represents the real spin system. Here we report a new J modulated method, constant-time INEPT CT-HSQC (CTi-CT-HSQC), to accurately measure one-bond scalar coupling constant and RDCs without strong coupling interference. In this method, changing the spacing between the two 180° pulses during a constant time INEPT period selectively modulates heteronuclear coupling in quantitative J fashion. Since the INEPT delays for measuring one-bond carbon-proton spectra are short compared to (3)J(HH), evolution due to (strong) (1)H-(1)H coupling is marginal. The resulting curve shape is practically independent of (1)H-(1)H coupling and only correlated to the heteronuclear coupling evolution. Consequently, an accurate (1)J(CH) can be measured even in the presence of strong coupling. We tested this method on N-acetyl-glucosamine and mannose whose apparent isotropic (1)J(CH) values are significantly affected by strong coupling with other methods. Agreement to within 0.5Hz or better is found between (1)J(CH) measured by this method and previously published simulation data. We further examined the strong coupling effects on RDC measurements and observed an error up to 100% for one bond RDCs using coupled HSQC in carbohydrates. We demonstrate that RDCs can be obtained with higher accuracy by CTi-CT-HSQC, which compensates the limitation of simulation method.

NMR profiling of biomolecules at natural abundance using 2D 1H–15N and 1H–13C multiplicity-separated (MS) HSQC spectra

2D NMR (1)H-X (X=(15)N or (13)C) HSQC spectra contain cross-peaks for all XHn moieties. Multiplicity-edited(1)H-(13)C HSQC pulse sequences generate opposite signs between peaks of CH(2) and CH/CH(3) at a cost of lower signal-to-noise due to the (13)C T(2) relaxation during an additional 1/(1)JCH period. Such CHn-editing experiments are useful in assignment of chemical shifts and have been successfully applied to small molecules and small proteins (e.g. ubiquitin) dissolved in deuterated solvents where, generally, peak overlap is minimal. By contrast, for larger biomolecules, peak overlap in 2D HSQC spectra is unavoidable and peaks with opposite phases cancel each other out in the edited spectra. However, there is an increasing need for using NMR to profile biomolecules at natural abundance dissolved in water (e.g., protein therapeutics) where NMR experiments beyond 2D are impractical. Therefore, the existing 2D multiplicity-edited HSQC methods must be improved to acquire data on nuclei other than (13)C (i.e.(15)N), to resolve more peaks, to reduce T(2) losses and to accommodate water suppression approaches. To meet these needs, a multiplicity-separated(1)H-X HSQC (MS-HSQC) experiment was developed and tested on 500 and 700 MHz NMR spectrometers equipped with room temperature probes using RNase A (14 kDa) and retroviral capsid (26 kDa) proteins dissolved in 95% H(2)O/5% D(2)O. In this pulse sequence, the 1/(1)JXH editing-period is incorporated in to the semi-constant time (semi-CT) X resonance chemical shift evolution period, which increases sensitivity, and importantly, the sum and the difference of the interleaved (1)J(XH)-active and the (1)J(XH)-inactive HSQC experiments yield two separate spectra for XH(2) and XH/XH(3). Furthermore we demonstrate improved water suppression using triple xyz-gradients instead of the more widely used z-gradient only water-suppression approach.

Transient hydrogen bonding in uniformly 13C,15N‐Labeled Carbohydrates in Water

We report NMR studies of transient hydrogen bonding in a polysaccharide (PS) dissolved in water without cosolvent at ambient temperature. The PS portion of the Escherichia coli O142 lipopolysaccharide is comprised of repeating pentasaccharide units of GalNAc (N-acetyl galactosamine), GlcNAc (N-acetyl glucosamine), and rhamnose in a 3:1:1 ratio, respectively. A 105-ns molecular dynamics (MD) simulation on one pentasaccharide repeat unit predicts transient inter-residue hydrogen bonds from the GalNAc NH groups in the PS. To investigate these predictions experimentally, the PS was uniformly ¹³C,¹⁵N enriched and the NH, carbonyl, C2, C4, and methyl resonances of the GalNAc and GlcNAc residues assigned using through-bond triple-resonance NMR experiments. Temperature dependence of amide NH chemical shifts and one-bond NH J couplings support that NH groups on two of the GalNAc residues are donors in transient hydrogen bonds. The remaining GalNAc and GlcNAc NHs do not appear to be donors from either temperature-dependent chemical shifts or one-bond NH J couplings. These results substantiate the presence of weak or partial hydrogen bonds in carbohydrates, and that MD simulations of repeating units in PSs provide insight into overall PS structure and dynamics.

NMR detection and characterization of sialylated glycoproteins and cell surface polysaccharides.

Few solution NMR pulse sequences exist that are explicitly designed to characterize carbohydrates (glycans). This is despite the essential role carbohydrate motifs play in cell–cell communication, microbial pathogenesis, autoimmune disease progression and cancer metastasis, and despite that fact that glycans, often shed to extra-cellular fluids, can be diagnostic of disease. Here we present a suite of two dimensional coherence experiments to measure three different correlations (H3–C2, H3–C1, and C1–C2) on sialic acids, a group of nine-carbon carbohydrates found on eukaryotic cell surfaces that often play a key role in disease processes. The chemical shifts of the H3, C2, and C1 nuclei of sialic acids are sensitive to carbohydrate linkage, linkage conformation, and ionization state of the C1 carboxylate. The experiments reported include rigorous filter elements to enable detection and characterization of isotopically labeled sialic acids with high sensitivity in living cells and crude isolates with minimal interference from unwanted signals arising from the ~1% 13C-natural abundance of cellular metabolites. Application is illustrated with detection of sialic acids on living cells, in unpurified mixtures, and at the terminus of the N-glycan on the 55xa0kDa immunoglobulin G Fc.

Single synonymous mutation in factor IX alters protein properties and underlies haemophilia B

Background Haemophilia B is caused by genetic aberrations in the F9 gene. The majority of these are non-synonymous mutations that alter the primary structure of blood coagulation factor IX (FIX). However, a synonymous mutation c.459G>A (Val107Val) was clinically reported to result in mild haemophilia B (FIX coagulant activity 15%–20% of normal). The F9 mRNA of these patients showed no skipping or retention of introns and/or change in mRNA levels, suggesting that mRNA integrity does not contribute to the origin of the disease in affected individuals. The aim of this study is to elucidate the molecular mechanisms that can explain disease manifestations in patients with this synonymous mutation. Methods We analyse the molecular mechanisms underlying the FIX deficiency through in silico analysis and reproducing the c.459G>A (Val107Val) mutation in stable cell lines. Conformation and non-conformation sensitive antibodies, limited trypsin digestion, activity assays for FIX, interaction with other proteins and post-translation modifications were used to evaluate the biophysical and biochemical consequences of the synonymous mutation. Results The Val107Val synonymous mutation in F9 was found to significantly diminish FIX expression. Our results suggest that this mutation slows FIX translation and affects its conformation resulting in decreased extracellular protein level. The altered conformation did not change the specific activity of the mutated protein. Conclusions The pathogenic basis for one synonymous mutation (Val107Val) in the F9 gene associated with haemophilia B was determined. A mechanistic understanding of this synonymous variant yields potential for guiding and developing future therapeutic treatments.