Structure determination of the Antp(C39 → S) homeodomain from nuclear magnetic resonance data in solution using a novel strategy for the structure calculation with the programs DIANA, CALIBA, HABAS and GLOMSA

doi:10.1016/0022-2836(91)90755-U

Journal of Molecular Biology

Volume 217, Issue 3, 5 February 1991, Pages 531-540

https://doi.org/10.1016/0022-2836(91)90755-U Get rights and content

Abstract

The structure of a mutant Antennapedia homeodomain, Antp(C39 → S), from Drosophila melanogaster was determined using a set of new programs introduced in the accompanying paper. An input dataset of 957 distance constraints and 171 dihedral angle constraints was collected using two-dimensional n.m.r. experiments with the ¹⁵N-labeled protein. The resulting high quality structure for Antp(C39 → S), with an average root-mean-square deviation of 0.53 Å between the backbone atoms of residues 7 to 59 in 20 energy-refined distance geometry structures and the mean structure, is nearly identical to the previously reported structure of the wild-type Antp homeodomain. The only significant difference is in the connection between helices III and IV, which was found to be less kinked than was indicated by the structure determination for Antp. The main emphasis of the presentation in this paper is on a detailed account of the practical use of a novel strategy for the computation of nuclear magnetic resonance structures of proteins with the combined use of the programs DIANA, CALIBA, HABAS and GLOMSA.

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The 13C and 15N chemical shifts were referenced indirectly (41). All NOEs were assigned manually and integrated nuclear Overhauser effect (NOE) peaks were calibrated and converted into distance restraints with the program CALIBA (42). In the final structural determination, the program CYANA-3.0 (43) was used.
Leishmania donovani possess two isoforms of Rab5 (Rab5a and Rab5b), which are involved in fluid phase and receptor-mediated endocytosis, respectively. We have characterized the solution structure and dynamics of a stabilized truncated LdRab5a mutant. For the purpose of NMR structure determination, protein stability was enhanced by systematically introducing various deletions and mutations. Deletion of hypervariable C-terminal and the 20 residues LdRab5a specific insert slightly enhanced the stability, which was further improved by C107S mutation. The final construct, truncated LdRab5a with C107S mutation, was found to be stable for longer durations at higher concentration, with an increase in melting temperature by 10°C. Solution structure of truncated LdRab5a shows the characteristic GTPase fold having nucleotide and effector binding sites. Orientation of switch I and switch II regions match well with that of guanosine 5'-(β, γ-imido)triphosphate (GppNHp)-bound human Rab5a, indicating that the truncated LdRab5a attains the canonical GTP bound state. However, the backbone dynamics of the P-loop, switch I, and switch II regions were slower than that observed for guanosine 5'-(β, γ-imido)triphosphate (GMPPNP)-bound H-Ras. This dynamic profile may further complement the residue-specific complementarity in determining the specificity of interaction with the effectors. In parallel, biophysical investigations revealed the urea induced unfolding of truncated LdRab5a to be a four-state process that involved two intermediates, I1 and I2. The maximal 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (Bis-ANS) binding was observed for I2 state, which was inferred to have molten globule like characteristics. Overall, the strategy presented would have significant impact for studying other Rab and small GTPase proteins by NMR spectroscopy.
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The GMF class of the ADF-H domain family proteins regulate actin dynamics by binding to the Arp2/3 complex and F-actin through their Site-1 and Site-2, respectively. CeGMF of C. elegans is analogous to GMFγ of human and mouse and is 138 amino acids in length.
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We have presented the first solution structure of GMF from C. elegans, which highlights the structural differences between the Site-2 of CeGMF and mammalian GMF isoforms. Further, we have seen the differences in structure, dynamics, and thermal stability of GMF and ADF/cofilin.
This study provides a useful insight to structural and dynamics factors that define the specificity of GMF towards Arp2/3 complex.
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On the basis of structure, dynamics, and biochemical activity, Twinstar can be taken to execute its biochemical role by facilitating directional growth and maintenance of length of actin filaments.
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Citation Excerpt :
For the less defined protein regions (like Ser 19–Lys 27 and Asn 64–Glu 68 calcium-binding loop fragments or the C-terminus of helix IV starting from Val 83) the dihedral angle constraints were not applied. Stereospecific assignments for 122 side chain chiral groups in each subunit were generated by the program GLOMSA (Guentert et al., 1991) that is included in the CYANA software. Before the final structure calculations, 184 constraints defined as rHN-O = 1.5–2.5 Å and rN-O = 2.5–3.5 Å for 92 hydrogen bonds were added based on geometric criteria.
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Journal of Molecular Biology

ArticleStructure determination of the Antp(C39 → S) homeodomain from nuclear magnetic resonance data in solution using a novel strategy for the structure calculation with the programs DIANA, CALIBA, HABAS and GLOMSA

Abstract

J. Mol. Graph

J. Mol. Biol

J. Mol. Biol

Biochim. Biophys. Acta

J. Magn. Reson

J. Mol. Biol

Cell

J. Mol. Biol

Cell

Biochim. Biophys. Acta

J. Mol. Biol

J. Mol. Biol

J. Mol. Graph

Article
Structure determination of the Antp(C39 → S) homeodomain from nuclear magnetic resonance data in solution using a novel strategy for the structure calculation with the programs DIANA, CALIBA, HABAS and GLOMSA