Chemical shift assignments and secondary structure of the Grb2 SH2 domain by heteronuclear NMR spectroscopy

Wang, Yu-Sen; Frederick, Anne F.; Senior, Mary M.; Lyons, Barbara; Black, Stuart; Kirschmeier, Paul T.; Perkins, Louise M.; Wilson, Oswald

doi:10.1007/bf00203819

Cited by 9 publications

(4 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The backbone amide signals of LBT-Grb2 containing Lu 3+ were assigned based on the assignments reported previously (Wang et al 1996; Thornton et al 1996; Ogura et al 2008). 1 H– 15 N HSQC spectra of the 15 N-labeled LBT-Grb2 were recorded in the presence of 1 equivalent of lanthanide ions: Lu 3+ , Tb 3+ , Dy 3+ , Er 3+ and Tm 3+ (Fig.…”

Section: Resultsmentioning

confidence: 99%

An NMR strategy for fragment-based ligand screening utilizing a paramagnetic lanthanide probe

et al. 2011

View full text Add to dashboard Cite

A nuclear magnetic resonance-based ligand screening strategy utilizing a paramagnetic lanthanide probe is presented. By fixing a paramagnetic lanthanide ion to a target protein, a pseudo-contact shift (PCS) and a paramagnetic relaxation enhancement (PRE) can be observed for both the target protein and its bound ligand. Based on PRE and PCS information, the bound ligand is then screened from the compound library and the structure of the ligand–protein complex is determined. PRE is an isotropic paramagnetic effect observed within 30 Å from the lanthanide ion, and is utilized for the ligand screening in the present study. PCS is an anisotropic paramagnetic effect providing long-range (~40 Å) distance and angular information on the observed nuclei relative to the paramagnetic lanthanide ion, and utilized for the structure determination of the ligand–protein complex. Since a two-point anchored lanthanide-binding peptide tag is utilized for fixing the lanthanide ion to the target protein, this screening method can be generally applied to non-metal-binding proteins. The usefulness of this strategy was demonstrated in the case of the growth factor receptor-bound protein 2 (Grb2) Src homology 2 (SH2) domain and its low- and high-affinity ligands.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-011-9566-5) contains supplementary material, which is available to authorized users.

show abstract

Section: Resultsmentioning

confidence: 99%

An NMR strategy for fragment-based ligand screening utilizing a paramagnetic lanthanide probe

et al. 2011

View full text Add to dashboard Cite

show abstract

“…Singer, L. E. Kay & J. D. Forman-Kay, manuscript in preparation). It is also noteworthy that CSI predictions of secondary structural elements in the Grb2 SH2 domain have also severely underestimated the length of the RB helix (Wang et al, 1996).…”

Section: Resultsmentioning

confidence: 99%

Global Folds of Highly Deuterated, Methyl-Protonated Proteins by Multidimensional NMR

1997

View full text Add to dashboard Cite

The development of 15N, 13C, 2H multidimensional NMR spectroscopy has facilitated the assignment of backbone and side chain resonances of proteins and protein complexes with molecular masses of over 30 kDa. The success of these methods has been achieved through the production of highly deuterated proteins; replacing carbon-bound protons with deuterons significantly improves the sensitivity of many of the experiments used in chemical shift assignment. Unfortunately, uniform deuteration also radically depletes the number of interproton distance restraints available for structure determination, degrading the quality of the resulting structures. Here we describe an approach for improving the precision and accuracy of global folds determined from highly deuterated proteins through the use of deuterated, selectively methyl-protonated samples. This labeling profile maintains the efficiency of triple-resonance NMR experiments while retaining a sufficient number of protons at locations where they can be used to establish NOE-based contacts between different elements of secondary structure. We evaluate how this deuteration scheme affects the sensitivity and resolution of experiments used to assign 15N, 13C, and 1H chemical shifts and interproton NOEs. This approach is tested experimentally on a 14 kDa SH2/phosphopeptide complex, and a global protein fold is obtained from a set of methyl−methyl, methyl−NH, and NH−NH distance restraints. We demonstrate that the inclusion of methyl−NH and methyl−methyl distance restraints greatly improves the precision and accuracy of structures relative to those generated with only NH−NH distance restraints. Finally, we examine the general applicability of this approach by determining the structures of several proteins with molecular masses of up to 40 kDa from simulated distance and dihedral angle restraint tables.

show abstract

“…During the review of this article, the chemical shift assignments and secondary structure of the Grb2 SH2 domain were reported (Wang et al, 1996)…”

Section: Note Added In Proofmentioning

confidence: 99%

Nuclear Magnetic Resonance Solution Structure of the Growth Factor Receptor-Bound Protein 2 Src Homology 2 Domain

Thornton¹,

Mueller²,

McConnell³

et al. 1996

Biochemistry

View full text Add to dashboard Cite

A family of NMR solution structures of the growth factor receptor-bound protein 2 (Grb2) SH2 domain has been determined by heteronuclear multidimensional NMR. Proton, nitrogen, and carbon chemical shift assignments have been made for the SH2 domain of Grb2. Assignments were made from a combination of homonuclear two-dimensional and 15N- and 13C-edited three-dimensional spectra at pH 6.2 and 298 K. Structure-induced proton and carbon secondary shifts were calculated and used to facilitate the spectral assignment process. NOE, scalar coupling, secondary chemical shift, and amide proton exchange data were used to characterize the secondary structural elements and hydrogen-bonding network in the Grb2 SH2 domain. The three-dimensional structure of the Grb2 SH2 domain was calculated using 1112 restraints obtained from NOE, coupling constant, and amide proton exchange data. The rmsd for the 24 calculated structures to the mean structure of the Grb2 SH2 domain was 0.75 A for backbone and 1.28 A for all heavy atoms. The three-dimensional fold of the Grb2 SH2 domain is similar to that observed for other SH2 domains and consists of two alpha-helical segments and eight beta-strands, six strands that make up two contiguous antiparallel beta-sheets, and two strands that form two short parallel beta-sheets. The structure of the phosphotyrosine binding pocket of Grb2 is similar to that observed for other SH2 domains. The hydrophobic binding pocket of Grb2 is similar to that observed for Src with the exception that tryptophan 121 of Grb2 occupies part of the pY+3 binding pocket. Structural implications for the Grb2 SH2 domain selectivity at the pY+2 and pY+3 sites are discussed.

show abstract

Chemical shift assignments and secondary structure of the Grb2 SH2 domain by heteronuclear NMR spectroscopy

Cited by 9 publications

References 42 publications

An NMR strategy for fragment-based ligand screening utilizing a paramagnetic lanthanide probe

An NMR strategy for fragment-based ligand screening utilizing a paramagnetic lanthanide probe

Global Folds of Highly Deuterated, Methyl-Protonated Proteins by Multidimensional NMR

Nuclear Magnetic Resonance Solution Structure of the Growth Factor Receptor-Bound Protein 2 Src Homology 2 Domain

Contact Info

Product

Resources

About