NMR Chemical Shift Mapping of SH2 Peptide Interactions

McKercher, Marissa A.; Wuttke, Deborah S.

doi:10.1007/978-1-4939-6762-9_15

Cited by 3 publications

(5 citation statements)

References 39 publications

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“…All 1 H– 15 N heteronuclear single-quantum coherence ( 1 H– 15 N HSQC) experiments were performed on 700 μM PLCC in NMR buffer [100 mM sodium phosphate (pH 6.4) and 500 μM DTT] with 10% D 2 O and 150 μM TSP as previously described . Briefly, unlabeled phosphopeptide was resuspended in NMR buffer to a concentration of 8.75 mM and titrated into PLCC in 0.25 molar ratio increments to a final protein/peptide ratio of 1/1.25.…”

Section: Methodsmentioning

confidence: 99%

“…All 1 H− 15 N heteronuclear single-quantum coherence ( 1 H− 15 N HSQC) experiments were performed on 700 μM PLCC in NMR buffer [100 mM sodium phosphate (pH 6.4) and 500 μM DTT] with 10% D 2 O and 150 μM TSP as previously described. 40 Briefly, unlabeled phosphopeptide was resuspended in NMR buffer to a concentration of 8.75 mM and titrated into PLCC in 0.25 molar ratio increments to a final protein/peptide ratio of 1/1.25. An additional titration point (bringing the molar excess of the peptide to 1.5) produced identical chemical shift locations and intensities, indicating full saturation had been achieved with a 1.25-fold molar excess of peptide.…”

Section: ■ Experimental Proceduresmentioning

confidence: 99%

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Multimodal Recognition of Diverse Peptides by the C-Terminal SH2 Domain of Phospholipase C-γ1 Protein

2017

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SH2 domains recognize phosphotyrosine (pY)-containing peptide ligands and play key roles in the regulation of receptor tyrosine kinase pathways. Each SH2 domain has individualized specificity, encoded in the amino acids neighboring the pY, for defined targets that convey their distinct functions. The C-terminal SH2 domain (PLCC) of the phospholipase C-γ1 full-length protein (PLCγ1) typically binds peptides containing small and hydrophobic amino acids adjacent to the pY, including a peptide derived from platelet-derived growth factor receptor B (PDGFRB) and an intraprotein recognition site (Y783 of PLCγ1) involved in the regulation of the protein's lipase activity. Remarkably, PLCC also recognizes unexpected peptides containing amino acids with polar or bulky side chains that deviate from this pattern. This versatility in recognition specificity may allow PLCγ1 to participate in diverse, previously unrecognized, signaling pathways in response to binding chemically dissimilar partners. We have used structural approaches, including nuclear magnetic resonance and X-ray crystallography, to elucidate the mechanisms of noncognate peptide binding to PLCC by ligands derived from receptor tyrosine kinase ErbB2 and from the insulin receptor. The high-resolution peptide-bound structures reveal that PLCC has a relatively static backbone but contains a chemically rich protein surface comprised of a combination of hydrophobic pockets and amino acids with charged side chains. We demonstrate that this expansive and chemically diverse PLCC interface, in addition to peptide conformational plasticity, permits PLCC to recognize specific noncognate peptide ligands with multimodal specificity.

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Section: Methodsmentioning

confidence: 99%

Section: ■ Experimental Proceduresmentioning

confidence: 99%

Multimodal Recognition of Diverse Peptides by the C-Terminal SH2 Domain of Phospholipase C-γ1 Protein

2017

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“…Typical protein yields were approximately 40 mg/L. For isotopically labeled protein, SH2B1 was grown in minimal media with ( 15 NH 4 ) 2 SO 4 , as well as C‐glucose when intended for carbon‐labeled experiments, and otherwise expressed and purified as described above.…”

Section: Methodsmentioning

confidence: 99%

“…1 H‐ 15 N‐HSQC experiments used for chemical shift mapping were collected on 250 μM of 1 H‐ 15 N‐labeled SH2B1 in NMR buffer as previously described . A high protein concentration (250 µM SH2B1) was used in order to achieve sufficient signal to noise to collect data in a short time (approximately 80 min per titration point).…”

Section: Methodsmentioning

confidence: 99%

Diversity in peptide recognition by the SH2 domain of SH2B1

2017

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SH2B1 is a multidomain protein that serves as a key adaptor to regulate numerous cellular events, such as insulin, leptin, and growth hormone signaling pathways. Many of these protein-protein interactions are mediated by the SH2 domain of SH2B1, which recognizes ligands containing a phosphorylated tyrosine (pY), including peptides derived from janus kinase 2, insulin receptor, and insulin receptor substrate-1 and -2. Specificity for the SH2 domain of SH2B1 is conferred in these ligands either by a hydrophobic or an acidic side chain at the +3 position C-terminal to the pY. This specificity for chemically disparate species suggests that SH2B1 relies on distinct thermodynamic or structural mechanisms to bind to peptides. Using binding and structural strategies, we have identified unique thermodynamic signatures for each peptide binding mode, and several SH2B1 residues, including K575 and R578, that play distinct roles in peptide binding. The high-resolution structure of the SH2 domain of SH2B1 further reveals conformationally plastic protein loops that may contribute to the ability of the protein to recognize dissimilar ligands. Together, numerous hydrophobic and electrostatic interactions, in addition to backbone conformational flexibility, permit the recognition of diverse peptides by SH2B1. An understanding of this expanded peptide recognition will allow for the identification of novel physiologically relevant SH2B1/peptide interactions, which can contribute to the design of obesity and diabetes pharmaceuticals to target the ligand-binding interface of SH2B1 with high specificity.

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“…Nuclear magnetic resonance (NMR) is a powerful tool in studying H-bonds in biomolecules [ 2 , 6 , 22 , 23 , 24 , 25 , 26 , 27 ]. Using parameters such as chemical shifts [ 28 , 29 ], J-coupling constants [ 30 , 31 , 32 ], and H-D exchange rates [ 33 , 34 ], it is possible to study not only individual hydrogen bonds’ strengths but also their couplings. In conjunction with computational simulations, 1 H NMR can reveal the H-bonding coupling within the active site of some enzymes, such as ketosteroid isomerase and photoactive yellow protein [ 35 , 36 ].…”

Section: Introductionmentioning

confidence: 99%

Detecting the Hydrogen Bond Cooperativity in a Protein β-Sheet by H/D Exchange

Chen

Wang

et al. 2022

IJMS

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The hydrogen bond (H-bond) cooperativity in the β-sheet of GB3 is investigated by a NMR hydrogen/deuterium (H/D) exchange method. It is shown that the weakening of one backbone N–H…O=C H-bond between two β-strands, β1 and β2, due to the exchange of NH to ND of the H-bond donor in β1, perturbs the chemical shift of 13Cα, 13Cβ, 1Hα, 1HN, and 15N of the H-bond acceptor and its following residue in β2. Quantum mechanical calculations suggest that the -H-bond chemical shift isotope effect is caused by the structural reorganization in response to the H-bond weakening. This structural reorganization perturbs four neighboring H-bonds, with three being weaker and one being stronger, indicating that three H-bonds are cooperative and one is anticooperative with the perturbed H-bond. The sign of the cooperativity depends on the relative position of the H-bonds. This H-bond cooperativity, which contributes to β-sheet stability overall, can be important for conformational coupling across the β-sheet.

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NMR Chemical Shift Mapping of SH2 Peptide Interactions

Cited by 3 publications

References 39 publications

Multimodal Recognition of Diverse Peptides by the C-Terminal SH2 Domain of Phospholipase C-γ1 Protein

Multimodal Recognition of Diverse Peptides by the C-Terminal SH2 Domain of Phospholipase C-γ1 Protein

Diversity in peptide recognition by the SH2 domain of SH2B1

Detecting the Hydrogen Bond Cooperativity in a Protein β-Sheet by H/D Exchange

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