Crystal structure of the GDP-bound GTPase domain of Rab5a from Leishmania donovani.

Eukaryotic Rab5s are highly conserved small GTPase-family proteins that are involved in the regulation of early endocytosis. Leishmania donovani Rab5a regulates the sorting of early endosomes that are involved in the uptake of essential nutrients through fluid-phase endocytosis. Here, the 1.80 Šresolution crystal structure of the N-terminal GTPase domain of L. donovani Rab5a in complex with GDP is presented. The crystal structure determination was enabled by the design of specific single-site mutations and two deletions that were made to stabilize the protein for previous NMR studies. The structure of LdRab5a shows the canonical GTPase fold, with a six-stranded central mixed ß-sheet surrounded by five α-helices. The positions of the Switch I and Switch II loops confirm an open conformation, as expected in the absence of the γ-phosphate. However, in comparison to other GTP-bound and GDP-bound homologous proteins, the Switch I region traces a unique disposition in LdRab5a. One magnesium ion is bound to the protein at the GTP-binding site. Molecular-dynamics simulations indicate that the GDP-bound structure exhibits higher stability than the apo structure. The GDP-bound LdRab5a structure presented here will aid in efforts to unravel its interactions with its regulators, including the guanine nucleotide-exchange factor, and will lay the foundation for a structure-based search for specific inhibitors.

Structural and Biophysical Characterization of Rab5a from Leishmania Donovani.

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'-(ß, 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'-(ß, 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.

Solution structure and dynamics of glia maturation factor from Caenorhabditis elegans.

BACKGROUND: 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. METHODS: We have characterized the solution structure and dynamics of CeGMF by solution NMR spectroscopy and its thermal stability by DSC. RESULTS: The solution structure of CeGMF shows canonical ADF-H fold with two additional ß-strands in the ß4-ß5 loop region. The Site-1 of CeGMF is well formed and residues of all three regions of Site-1 show dynamic flexibility. However, the ß4-ß5 loop of Site-2 is less inclined towards the C-terminal, as the latter is truncated by four residues in comparison to GMF isoforms of human and mouse. Regions of Site-2 show motions on ns-ps timescale, but dynamic flexibility of ß4-ß5 loop is low in comparison to corresponding F-loop region of ADF/cofilin UNC-60B. A general difference in packing of α3 and α1 between GMF and ADF/cofilins was noticed. Additionally, thermal stability of CeGMF was significantly higher than its ADF/cofilin homologs. CONCLUSION: 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. GENERAL SIGNIFICANCE: This study provides a useful insight to structural and dynamics factors that define the specificity of GMF towards Arp2/3 complex.

Structure, dynamics, and biochemical characterization of ADF/cofilin Twinstar from Drosophilamelanogaster.

BACKGROUND: Twinstar is an ADF/cofilin family protein, which is expressed by the tsr gene in Drosophila melanogaster. Twinstar is one of the main regulators of actin cytoskeleton remodelling and is essential for vital cellular processes like cytokinesis and endocytosis. METHODS: We have characterized the structure and dynamics of Twinstar by solution NMR spectroscopy, the interaction of Twinstar with rabbit muscle actin by ITC, and biochemical activities of Twinstar through different biochemical assays using fluorescence spectroscopy and ultra-centrifugation. RESULTS: The solution structure of Twinstar shows characteristic ADF-H fold with well-formed G/F-site and F-site for interaction with actin. The structure possesses an extended F-loop, which is rigid at the base, but flexible towards its apical region. Twinstar shares similar dynamics for the G/F-site with C. elegans homologs, UNC-60A and UNC-60B. However, the dynamics of its F-loop are different from its C. elegans homologs. Twinstar shows strong affinity for ADP-G-Actin and ATP-G-Actin with Kds of ~7.6 nM and ~0.4 µM, respectively. It shows mild F-actin depolymerizing activity and stable interaction with F-actin with a Kd of ~5.0 µM. It inhibits the rate of the nucleotide exchange in a dose dependent manner. CONCLUSION: 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. GENERAL SIGNIFICANCE: This study characterizes the structure, backbone dynamics, and biochemical activities of Twinstar of Drosophila, which provides an insight into the regulation of actin dynamics in the member of phylum insecta.

Solution structures and dynamics of ADF/cofilins UNC-60A and UNC-60B from Caenorhabditis elegans.

The nematode Caenorhabditis elegans has two ADF (actin-depolymerizing factor)/cofilin isoforms, UNC-60A and UNC-60B, which are expressed by the unc60 gene by alternative splicing. UNC-60A has higher activity to cause net depolymerization, and to inhibit polymerization, than UNC-60B. UNC-60B, on the other hand, shows much stronger severing activity than UNC-60A. To understand the structural basis of their functional differences, we have determined the solution structures of UNC-60A and UNC-60B proteins and characterized their backbone dynamics. Both UNC-60A and UNC-60B show a conserved ADF/cofilin fold. The G-actin (globular actin)-binding regions of the two proteins are structurally and dynamically conserved. Accordingly, UNC-60A and UNC-60B individually bind to rabbit muscle ADP-G-actin with high affinities, with Kd values of 32.25 nM and 8.62 nM respectively. The primary differences between these strong and weak severing proteins were observed in the orientation and dynamics of the F-actin (filamentous actin)-binding loop (F-loop). In the strong severing activity isoform UNC-60B, the orientation of the F-loop was towards the recently identified F-loop-binding region on F-actin, and the F-loop was relatively more flexible with 14 residues showing motions on a nanosecond-picosecond timescale. In contrast, in the weak severing protein isoform UNC-60A, the orientation of the F-loop was away from the F-loop-binding region and inclined towards its own C-terminal and strand ß6. It was also relatively less flexible with only five residues showing motions on a nanosecond-picosecond timescale. These differences in structure and dynamics seem to directly correlate with the differential F-actin site-binding and severing properties of UNC-60A and UNC-60B, and other related ADF/cofilin proteins.