Processing of heteronuclear NMR relaxation data with the new software DASHA

Orekhov, Vladislav Yu.; Nolde, Dmitry E.; Golovanov, Alexander P.; Korzhnev, Dmitry M.; Arseniev, Alexander S.

doi:10.1007/bf03162365

Cited by 79 publications

(68 citation statements)

References 18 publications

(33 reference statements)

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“…NMR data were processed using NMRPipe (41), and analyzed using NMRView (42). The selection of ordered, nonexchanging resonances from the nSH2 domain, and the estimation and error analysis of rotational correlation times were done using DASHA (43). HYDRONMR (34) was used to calculate the expected rotational correlation times and relaxation rates for the individual and combined p85ni domains.…”

Section: Methodsmentioning

confidence: 99%

Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110α and are disrupted in oncogenic p85 mutants

Shekar

Flinn

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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We previously proposed a model of Class IA PI3K regulation in which p85 inhibition of p110␣ requires (i) an inhibitory contact between the p85 nSH2 domain and the p110␣ helical domain, and (ii) a contact between the p85 nSH2 and iSH2 domains that orients the nSH2 so as to inhibit p110␣. We proposed that oncogenic truncations of p85 fail to inhibit p110 due to a loss of the iSH2-nSH2 contact. However, we now find that within the context of a minimal regulatory fragment of p85 (the nSH2-iSH2 fragment, termed p85ni), the nSH2 domain rotates much more freely ( c Ϸ12.7 ns) than it could if it were interacting rigidly with the iSH2 domain. These data are not compatible with our previous model. We therefore tested an alternative model in which oncogenic p85 truncations destabilize an interface between the p110␣ C2 domain (residue N345) and the p85 iSH2 domain (residues D560 and N564). p85ni-D560K/N564K shows reduced inhibition of p110␣, similar to the truncated p85ni-572 STOP . Conversely, wild-type p85ni poorly inhibits p110␣N345K. Strikingly, the p110␣N345K mutant is inhibited to the same extent by the wild-type or truncated p85ni, suggesting that mutation of p110␣-N345 is not additive with the p85ni-572 STOP mutation. Similarly, the D560K/N564K mutation is not additive with the p85ni-572 STOP mutant for downstream signaling or cellular transformation. Thus, our data suggests that mutations at the C2-iSH2 domain contact and truncations of the iSH2 domain, which are found in human tumors, both act by disrupting the C2-iSH2 domain interface.cancer ͉ glioblastoma ͉ phosphoinositide 3-kinase ͉ PIK3CA P I 3-kinases are important cellular regulators of growth, survival, and motility, and deregulation of PI 3-kinase signaling contributes to cancer and other human diseases (1). Class IA PI 3-kinases, which produce PI[3,4,5]P3 in intact cells (2), are obligate heterodimers of a regulatory subunit (p85␣, p85␤, p55␣, p50␣, or p55␥) and a catalytic subunit (p110␣, p110␤, or p110␦) (reviewed in ref.3). The regulatory subunits have two major functions: they stabilize the catalytic subunits against thermal denaturation, and they maintain the catalytic subunit in an inhibited, low activity state (4, 5).p85 and p110 are both multidomain proteins that bind to each other and to upstream activators such as Rac and Cdc42, Ras, and tyrosine phosphorylated receptors and adapters (reviewed in ref. 6). p85 contains an SH3 domain, a Rac/Cdc42-binding domain homologous to a GAP domain in the BCR gene product, and two SH2 domains that flank an antiparallel coiled coil domain (the iSH2 domain). While NMR, EPR, and crystal structures have been obtained for the individual domains (7-15), there are currently no structures that define how these domains are arranged in space. The p110␣ catalytic subunit has been better defined, with structures of the N-terminal adapter-binding domain (ABD) or the entire p110␣ bound to the coiled coil (iSH2) domain of p85 (15,16). Like the related Class IB catalytic subunit p110␥ (17), p110␣ contains Ras-binding, C2, ...

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Section: Methodsmentioning

confidence: 99%

Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110α and are disrupted in oncogenic p85 mutants

Shekar

Flinn

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…The rotation di¡usion tensor of Zrv-IIB was assessed by hydrodynamic calculations using the beads model approximation [20,21,24] based on the NMR structure of Zrv-IIB [8]. The calculations were performed with the DIFFC program [21].…”

Section: Hydrodynamic Calculationsmentioning

confidence: 99%

“…The calculations were performed with the DIFFC program [21]. Each residue of Zrv-IIB was represented by a bead of 3.5 A î radius centered at the position of its C K atom.…”

Section: Hydrodynamic Calculationsmentioning

confidence: 99%

Backbone dynamics of the channel‐forming antibiotic zervamicin IIB studied by ¹⁵N NMR relaxation

et al. 2001

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The backbone dynamics of the channel-forming peptide antibiotic zervamicin IIB (Zrv-IIB) in methanol were studied by 15 N nuclear magnetic resonance relaxation measurements at 11.7, 14.1 and 18.8 T magnetic fields. The anisotropic overall rotation of the peptide was characterized based on 15 N relaxation data and by hydrodynamic calculations.`Model-free' analysis of the relaxation data showed that the peptide is fairly rigid on a sub-nanosecond time-scale. The residues from the polar side of Zrv-IIB helix are involved in micro^millisecond time-scale conformational exchange. The conformational exchange observed might indicate intramolecular processes or specific intermolecular interactions of potential relevance to Zrv-IIB ion channel formation. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

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“…This method has been used successfully to calculate translational as well as rotational diffusion tensors of proteins (46,(69)(70)(71)(72)(73). In this method, the protein is modeled as a collection of point sources of Page 23 of 32 Figure S1…”

Section: Protein Hydrodynamic Calculationsmentioning

confidence: 99%

Temperature dependence of protein-hydration hydrodynamics by molecular dynamics simulations

Lau¹,

Krishnan²

2007

Biophysical Chemistry

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Processing of heteronuclear NMR relaxation data with the new software DASHA

Cited by 79 publications

References 18 publications

Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110α and are disrupted in oncogenic p85 mutants

Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110α and are disrupted in oncogenic p85 mutants

Backbone dynamics of the channel‐forming antibiotic zervamicin IIB studied by ¹⁵N NMR relaxation

Temperature dependence of protein-hydration hydrodynamics by molecular dynamics simulations

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Processing of heteronuclear NMR relaxation data with the new software DASHA

Cited by 79 publications

References 18 publications

Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110α and are disrupted in oncogenic p85 mutants

Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110α and are disrupted in oncogenic p85 mutants

Backbone dynamics of the channel‐forming antibiotic zervamicin IIB studied by 15N NMR relaxation

Temperature dependence of protein-hydration hydrodynamics by molecular dynamics simulations

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Backbone dynamics of the channel‐forming antibiotic zervamicin IIB studied by ¹⁵N NMR relaxation