Ligand Specificity Modulated by Prolyl Imide Bond Cis/Trans Isomerization in the Itk SH2 Domain:  A Quantitative NMR Study

Breheny, Patrick; Laederach, Alain; Fulton, D. Bruce; Andreotti, Amy H.

doi:10.1021/ja0375380

Cited by 41 publications

(58 citation statements)

References 6 publications

(7 reference statements)

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“…This high degree of sequence conservation strongly suggests an evolutionarily conserved role of proline isomerization in AHSP. Other proteins that have similar proline-mediated conformational exchange have been reported to show different ligand specificity for both conformers (31,33,34). The differences in the conformation of loop 1 between the two forms of the wildtype AHSP are big enough for this also to be the case with AHSP.…”

Section: Discussionmentioning

confidence: 99%

NMR Structure of the α-Hemoglobin Stabilizing Protein

Santiveri

Pérez‐Cañadillas

Vadivelu

et al. 2004

Journal of Biological Chemistry

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The structure of ␣-hemoglobin stabilizing protein (AHSP), a molecular chaperone for free ␣-hemoglobin, has been determined using NMR spectroscopy. The protein native state shows conformational heterogeneity attributable to the isomerization of the peptide bond preceding a conserved proline residue. The two equally populated cis and trans forms both adopt an elongated antiparallel three ␣-helix bundle fold but display major differences in the loop between the first two helices and at the C terminus of helix 3. Proline to alanine single point mutation of the residue Pro-30 prevents the cis/ trans isomerization. The structure of the P30A mutant is similar to the structure of the trans form of AHSP in the loop 1 region. Both the wild-type AHSP and the P30A mutant bind to ␣-hemoglobin, and the wild-type conformational heterogeneity is quenched upon complex formation, suggesting that just one conformation is the active form. Changes in chemical shift observed upon complex formation identify a binding interface comprising the C terminus of helix 1, the loop 1, and the N terminus of helix 2, with the exposed residues Phe-47 and Tyr-51 being attractive targets for molecular recognition. The characteristics of this interface suggest that AHSP binds at the intradimer ␣ 1 ␤ 1 interface in tetrameric HbA.␣-Hemoglobin stabilizing protein (AHSP), 1 previously named EDRF (erythroid differentiation-related factor), was originally identified as a protein that showed a decrease in expression in the hematopoietic tissues of rodents and cattle infected with transmissible spongiform encephalopathies (1). EDRF was renamed AHSP after it was found that it binds specifically to ␣-hemoglobin in vitro (2). AHSP knock-out mice show erythrocyte abnormalities similar to those observed in ␤-thalassemia (2), and loss of AHSP aggravates the ␤-thalassemia phenotype in mice (38). ␤-Thalassemia is characterized by a reduction in the synthesis of ␤-globin chains. Consequently, the ␣-chains are in excess, and some of them precipitate, leading to defective red blood cells that determine the pathophysiology of ␤-thalassemia syndromes. The in vivo and in vitro data therefore both suggest that AHSP acts as a molecular chaperone for free ␣-hemoglobin (2, 3).Both AHSP and ␣-hemoglobin are monomeric in solution and bind each other with an association constant of 1 ϫ 10 7 M Ϫ1forming a complex with a 1:1 stoichiometry (4). The aim of the present study was to structurally characterize the AHSP-␣-hemoglobin interaction to understand the molecular basis of the AHSP function and its role as a hemoglobin chaperone. We have demonstrated by NMR that the AHSP native state is conformationally heterogeneous in solution and have identified the dynamic source of this behavior as a cis/trans isomerization of residue Pro-30. The slow time scale of this phenomenon allowed us to determine the high resolution three-dimensional structure of the two conformations of the wild-type AHSP under conditions where both conformations are equally populated. We were able to quench the c...

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Section: Discussionmentioning

confidence: 99%

NMR Structure of the α-Hemoglobin Stabilizing Protein

Santiveri

Pérez‐Cañadillas

Vadivelu

et al. 2004

Journal of Biological Chemistry

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“…26 Alternatively, conformational states at Pro55 could influence the conformation of the loop region has an enzymatic activity of ca 46% with respect to the native state. (residues [43][44][45][46], which is involved in the recognition of the substrate guanosyl residue. 34 In the literature, an increasing number of examples report on cis/trans-isomerism-mediated conformational heterogeneity in the native states of proteins.…”

Section: Discussionmentioning

confidence: 99%

“…46 Conformation at Pro287 decides whether the binding of a phosphopeptide ligand or intermolecular association to a Src homology 3 domain is favored. In case of interleukin-2 tyrosine kinase Src homology 2 domain, the binding site of the physiological ligands encompasses the proline residue responsible for the conformational heterogeneity, although long-range structural effects have been observed in heteronuclear single quantum coherence NMR spectra.…”

Section: Discussionmentioning

confidence: 99%

Bioactivity of Folding Intermediates Studied by the Recovery of Enzymatic Activity during Refolding

Aumüller

Fischer

2008

Journal of Molecular Biology

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“…This switch regulates substrate recognition (Brazin et al, 2000). Cis-trans isomerization of a single prolyl-imide bond (D286-P287) within the SH2 domain influenced substrate recognition (Breheny et al, 2003;Mallis et al, 2002). In Btk the corresponding protein has only trans conformation (Huang et al, 2006).…”

Section: The Function Of Individual Domains In Tfksmentioning

confidence: 99%