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1vkk

    Table of contents
    1. 1. Protein Summary
    2. 2. Ligand Summary
    3. 3. References

    Title Crystal structure of Glia maturation factor-gamma (GMFG) from Mus musculus at 1.50 A resolution. To be published
    Site JCSG
    PDB Id 1vkk Target Id 354641
    Molecular Characteristics
    Source Mus musculus
    Alias Ids TPS1337,15079298, 89583, 89787, 289597, 289509, 289421, 289323, 89842 Molecular Weight 16747.48 Da.
    Residues 142 Isoelectric Point 5.57
    Sequence msdslvvcevdpelketlrkfrfrketnnaaiimkvdkdrqmvvledelqnispeelklelperqprfv vysykyvhddgrvsyplcfifsspvgckpeqqmmyagsknrlvqtaeltkvfeirttddltetwlkekl affr
      BLAST   FFAS

    Structure Determination
    Method XRAY Chains 1
    Resolution (Å) 1.35 Rfree 0.19411
    Matthews' coefficent 1.81 Rfactor 0.1581
    Waters 183 Solvent Content 31.56

    Ligand Information
    Ligands
    Metals

    Jmol

     
    Google Scholar output for 1vkk
    1. Domain definition and target classification for CASP6
    M Tress, CH Tai, G Wang, I Ezkurdia - PROTEINS: , 2005 - Wiley Online Library
     
    2. Fast and accurate algorithms for protein side-chain packing
    J Xu, B Berger - Journal of the ACM (JACM), 2006 - dl.acm.org
     
    3. The JCSG MR pipeline: optimized alignments, multiple models and parallel searches
    R Schwarzenbacher, A Godzik - Section D: Biological , 2007 - scripts.iucr.org
     
    4. Alternate states of proteins revealed by detailed energy landscape mapping
    MD Tyka, DA Keedy, I Andr, F DiMaio, Y Song - Journal of molecular , 2011 - Elsevier
     
    5. Autoindexing with outlier rejection and identification of superimposed lattices
    NK Sauter, BK Poon - Journal of Applied Crystallography, 2010 - scripts.iucr.org
     
    6. NMR solution structures of actin depolymerizing factor homology domains
    AK Goroncy, S Koshiba, N Tochio, T Tomizawa - Protein , 2009 - Wiley Online Library
     
    7. Protein structure prediction based on the sliced lattice model
    CC Wang, CB Yang, HY Ann - 2008 Conference on , 2005 - etd.lib.nsysu.edu.tw
     
    8. Prediction of Protein Backbone Based on the Sliced Lattice Model
    CC Wang, CB Yang, HY Ann, HY Chang - 2008 - csie.npu.edu.tw
     
    9. Protein loop prediction by fragment assembly
    Z Liu - Masters Abstracts International, 2007 - el.trc.gov.om
     

    Protein Summary

    Glia maturation factors (GMFs) are small (MW ~17 kDa) proteins implicated in the development of the nervous system, angiogenesis and immune function. GMFs bind actin and show remote sequence similarity to the ADF/cofilin family. However, distinct functionalities and regulatory mechanisms indicate a singular role for GMFs compared to all ADF/cofilin proteins described to date. The structure of GMFG, a GMF expressed in the blood and immune systems, shows similarity to the ADF-H domain shared by ADF/cofilins, twinfilins, and Abp1/drebrins. However, actin binding regions show only partial conservation in GMFs while the ADF/cofilin regulatory interface involved in phosphoinositide interactions is absent entirely. An exposed loop proximal to a β-hairpin critical for binding to F-actin, adopts a conformation unique to GMFs and suggests a model for regulation of GMF binding via phosphorylation. Additional differences in the stabilization of the actin-binding helix may account for the distinct cellular roles and biochemical activities that distinguish GMFs from other members of the ADF/cofilin family.

        

    GMFs adopt the typical actin-depolymerizing factor homology (ADF-H) fold found in the ADF/cofilin, twinfilin, and drebin/Abp1 families of actin-binding proteins (Lappalainen 1998) . The ADF-H fold consists of a central six-stranded mixed β-sheet (β1-β4 and β7-β8 in Fig. 1A) flanked by two pairs of α-helices (α1 and α3, α2 and α5 in Fig. 1A). Helices α1-α4 run parallel to the central β-sheet while α5 packs perpendicular to strands β4-β7. Another conserved characteristic of the ADF-H fold shared by GMFs is the kink in the middle of helix α3 (Bowman 2000), a helix implicated in actin-binding. The hydrophobic core of the crystal structure of GMFG, determined using PISA, comprises residues Ala30, Ala31, Ile32, Met34, Leu57, Pro66, Phe68, Val69, Tyr71, Cys87, Phe88, Ile89, Ser92, Leu134 and Leu138. Flexible regions of the protein, identified by elevated temperature factors, include the N- and C-terminal residues (strand β1, last helical turn of helix H5), the first and last helical turns of helix H3, and loops H1-β2 and H3-β8.

     

    The structures of mouse GMFB and GMFG have also been determined using triple resonance NMR techniques. A structural superposition of the crystal structure of GMFG (pdb id: 1vkk) with the lowest energy NMR structures of GMFB (pdb id: 1v6f) and GMFG (pdb id: 1wfs) reveals all three structures to be essentially identical with an r.m.s.d. of 1.1 and 1.0 Å over 131 and 132 residues respectively (Fig. 1B). The main differences reside in the N- and C-termini, which in the NMR structures contain modified sequences, and in the conformation of the β5-β6 hairpin. At the N-terminus, a GS-rich heptapeptide replaces the N-terminal methionine while at the C-terminus, the last four residues are similarly replaced, producing a slight change in the orientation of the C-terminal helix. These modifications allow visualization of the N-terminal residues, which are disordered in the crystal structure.

         

    Fig1_take2.png

    Figure 1. Overall structure of GMF. (A) Stereo ribbon diagram of the crystal structure of GMFG. Spectral coloring is from blue (N-terminus) to red (C-terminus). Secondary structural elements are indicated. (B) Stereo ribbon diagram of a superposition of the crystal structure of GMFG (gray, PDB id: 1vkk) with the solution structures of GMFB (blue, PDB id: 1v6f) and GMFG (magenta, PDB id: 1wfs).

         

    Sequence and structural similarities between GMFs and cofilin have been previously reported (Ikeda 2006, Gorbatyuk 2006, Paavilainen 2007).  A FATCAT  database search identifies several proteins from the ADF/cofilin family with significant structural similarities to GMFs. Yeast cofilin (PDB accession code: 1cfy), has the strongest similarity with an r.m.s.d. of 1.4 Å over 135 residues and a sequence identity of 16%, with human cofilin (PDB accession code: 1q8g) sharing similar values (r.m.s.d. of 1.9 Å over 136 residues, 15% sequence identity). Similar values are obtained for other ADF-H domains, including plant ADF, twinfilin and  drebrin and coactosin-like proteins. A structure-based alignment of the top vertebrate hits is shown in Figure 2A and a structural superposition of GMFG and a range of ADF-H domains is shown in Figure 2.

     

    Actin-binding helix
    Mutation of two highly conserved tyrosines (Tyr64 and Tyr101 in yeast cofilin) to phenylalanine decreases ADF/cofilin affinity for actin and has led to the suggestion that these residues orient and stabilize the actin-binding α3-helix by hydrogen-bonding via their hydroxyl groups (Bowman 2000). In GMFs, the corresponding residues are Phe68 and Tyr104, suggesting a weakening in the interaction with actin for GMFs. A similar substitution is encountered in coactosin (Hellman 2004) and may account for distinct stabilization mechanisms between close and distant homologs of the ADF/cofilin family.

     

    ADF-H G-actin-binding mode not shared by GMFs
    The recent elucidation of the structure of the ADF-H domain of twinfilin bound to ATP-G-actin (Paavilainen 2008), provides a structural model of the ADF-H G-actin-bound state and allows for an analysis of the G-actin:ADF-H binding interface. Binding occurs in a groove between actin subdomains 1 and 3 and the interface implicates three regions in ADF-H, with interacting residues highly conserved between ADF/cofilins and twinfilin (Paavilainen 2008).


    Comparison with of the ADF-H:G-actin complex with GMF indicates  that GMFs do not share the same binding interface as ADF-H. The first interacting region, implicates the N-terminal extension of the ADF-H domain (Gln176 in twinfilin). In other members of the ADF-H family, this residue conserves its hydrophilic character allowing hydrogen-bonding with the actin C-terminus. This region is disordered in GMFG. However, structure-based alignment indicates a hydrophobic substitution (Val6 in GMFG). In addition, N-terminal serine phosphorylation has been shown to have opposite effects in GMFG and ADF/cofilins (Ikeda 2006), suggesting a different interacting interface along the GMF N-terminus. In the second interacting region, two strictly conserved basic residues at the start of the actin-interacting helix α3 that form hydrogen bonds with the two actin sub-domains (Arg267, Arg269 in twinfilin) are in GMFs replaced by shorter-chain amino-acids carrying a negative charge (Glu99, Gln101) (Fig. 2A). Finally, residues interacting via salt-bridges or hydrogen bonds in the β-strand following the central helix (Lys294, Glu296 in twinfilin) are in GMF replaced by small hydrophilic and hydrophobic residues respectively (Thr119, Val121) (Fig. 2A).


    No direct interaction of GMFs with G-actin has been demonstrated to date. Because the structures of ATP- and ADP-G-actin are very similar to each other (Otterbein 2001, Rould 2006), we conclude that GMFs do not bind to G-actin in the same manner as ADF-H. Further experiments will be required to determine whether GMFs bind G-actin through a novel interface or whether their interaction is limited to actin filaments.

     

    (A)

    Fig2_GMFG.png

     

    (B)

    loop.png

    Figure 2. Comparison of GMF with other members of the ADF-H family. (A) Structure-based sequence alignment of GMFs and members of the ADF-H family. Mouse GMFG (PDB id: 1vkk), mouse GMFB (PDB id: 1v6f), mouse twinfilin-1 (PDB id: 1m4j), human twinfilin-2 (PDB id: 2vac), human cofilin (PDB id: 1q8g), chick cofilin (PDB id: 1tvj). The alignment was carried out using Expresso. Numbering and secondary structure refer to mouse GMFG as determined in this study. Percent identity (%id) and r.m.s.d. for optimal backbone superposition of each structure onto mouse GMFG are given at the end of each sequence. Regions critical for ADF-H interaction with both G- and F-actin (Ojala 2001, Pope 2004) are shaded in green. Regions determined by mutagenesis (Ono 2001) to be critical for binding to F-actin but not G-actin are shaded in green. The ADF-H basic cluster that has been proposed to bind acidic head groups (Gorbatyuk 2006) is high-lighted in red. (B) Stereo ribbon view of the distinct conformation observed in the GMF α1-β2 loop as compared to all other ADF-H family members. The superimposed structures are mouse GMFG (PDB id: 1vkk, in blue), C-terminal domain of mouse twinfilin (PDB id: 3daw, in magenta), yeast cofilin (PDB id: 1cfy, in gray), actin-depolymerizing fator from Arabidopsis thaliana (PDB id: 1f7s, in green), mouse coactosin (PDB id: 1wm4, in yellow), actophorin from Acantamoeba polyphaga (PDB id: 1cnu, in cyan).

         

    ADF-H phospholipid binding sites are absent in GMFs 

    The activity of ADF/cofilins in cells is regulated by a number of different mechanisms, including phosphorylation, binding to acidic phospholipids and pH. Acidic phospholipids, such as PIP2, have been shown to inhibit the activity of ADF/cofilins (Yonezawa 1990) and down-regulate the actin-sequestering activity of twinfilin in vitro (Palmgren 2001). PIP2 binding sites on cofilin have been mapped by both mutagenesis (Ojala 2001) and NMR (Pope 2004, Gorbatyuk 2006). To date, no similar studies have been reported for GMFs. Although there is no residue overlap between the two mapping methods, a structure-based alignment with GMFG shows that neither mapped region exhibits any significant similarity to GMFG (Fig. 2). In addition, the region C-terminal to α5 that in cofilin has also been mapped to bind PIP2 (Gorbatyuk 2006), is absent in GMFG while a basic cluster (cofilin residues Lys125-Lys127) that has been proposed to act as an alternate docking site for phosphoinositide head groups (Gorbatyuk 2006) is not present in GMFG (residues Val112-Thr114) (Fig. 2A). We conclude that GMFs do not bind acidic phospholipids through the regions mapped on ADF-H. Further experiments will be required to determine whether GMFs employ a novel mode of binding to phospholipids or whether such an interaction is absent entirely.

     

    ADF/cofilin pH-sensitivity is absent in GMFs


    The activities of ADF/cofilins also show pH-dependent regulation with actin fragmentation accelerated at basic pH (Bernstein 2000). In the case of human cofilin, pH-dependence has been suggested to be mainly dependent on a single, salt-bridged histidine residue (His133 in chick cofilin) (Pope 2004), strongly conserved in vertebrate ADF/cofilins, that stabilizes the F-actin binding region in both proteins. Additional modulation of cofilin-actin interaction has been proposed to arise from conserved histidines on actin located proximal to a model of the cofilin-actin binding site (McGough 1997).


    No pH regulation has been demonstrated for GMFs. In the GMFG structure, His133 is replaced by Lys119, and the acidic residue implicated in the regulating salt-bridge (Asp98 in cofilin, Glu98 in ADF) is in GMFG replaced by Pro85. In the same region of the molecule, located on the β5-β6 hairpin, Ser83, a residue that has been shown to be phosphorylated by PKA (Zaheer 1997), lies within 7 Å from Lys119 (Fig. 3). Lysine-phosphoserine salt-bridges are known to afford considerable stability to protein secondary structures (Errington 2005). At a distance of 4 Å from Ser83, the C-terminal arginine (Arg142) could provide a second coordinating residue for the phosphate group. The single, highly conserved histidine present in GMFs (His77) is not within salt-bridging distance of any acidic residue but in a protonated state might serve as an third interacting partner for phosphorylated Ser83 (Fig. 3). Thus, the GMFG structure supports the hypothesis that phosphorylation of Ser83 could replace the pH-regulation encountered in ADF/cofilins as a mechanism for maintaining the integrity of the F-actin binding site.
     

    LoopCloseUp.png

     

    Figure 3. Close-up of the GMF α1-β2 loop. Residues subject to post-translational modification and proposed to stabilize the interaction with actin are indicated in ball-and-stick and labeled.

     

    A loop conformation unique to GMFs suggests a novel mode of regulation and actin-binding


    Phosphorylation sites between GMFB and GMFG are conserved. N-terminal phosphorylation (Ser2, Ser4) of GMFG has been shown to affect cytoskeletal organization by up-regulating association to F-actin and Arp2/3 complex (Ikeda 2006), an effect opposite to that observed for ADF/cofilins (see Yonezawa 1990 for review). GMFB phosphorylation of Thr27, Ser53, Ser72 and Tyr84 by PKA has been suggested to serve a pro-apoptotic role (Zaheer 1996) although the functional relevance of these post-translational modifications with respect to cytoskeletal organization remains unclear.


    Within the ADF-H protein family, the β5-β6 hairpin (Fig. 1A) adopts different orientations (Fedorov 1997, Paavilainen 2002, Quintero-Monzon 2005), translating to distinct modes of actin filament binding (Ono 2001Quintero-Monzon 2005). For example, an orientation of this hairpin in the N-terminal domain of twinfilin towards the actin filament, causes a steric clash explaining the preference of this domain for monomeric versus filamentous actin (Paavilainen 2007). In GMFG, the β5-β6 hairpin adopts an orientation shared by the C-terminal domain of twinfilin, a domain that interacts with the sides of actin filaments like ADF/cofilins (Paavilainen 2007). However, as discussed above, GMFs do not share the same modes of regulation as ADF/cofilins and twinfilin.


    The differences in regulation of actin-binding between ADF/cofilins and GMFs could, at least partially, reside in the α1-β2 loop. In GMFs, the α1-β2 loop adopts a unique conformation not encountered in any other member of the ADF-H family, including distant homologs such as actophorin and coactosin (Fig. 2B). This conformation is not the result of lattice contacts in the GMFG crystal structure and is identical to the conformation observed in the two GFM structures determined by NMR (Fig. 1B). Hydrogen bonds involving a strictly conserved asparagine, provide main-chain hydrogen bonds both with the β4-β5 loop (Asn29-Lys74) and the β3-α2 loop (Asn29-Asn51). Additional hydrogen bonds with the β4-β5 loop (Ala30-Ser72) and within the α1-β2 loop (Arg24-Asn28) serve to stabilize the α1-β2 loop and bring it into proximity of the β5-β6 hairpin.


    In the GMFG structure, Thr27 is located on the β5-β6 hairpin, and at a distance of 5 Å from Tyr84, in the α1-β2 loop (Fig. 3). Along with Ser72 and Ser83, these four residues form a contiguous surface on the GMFG molecule. A superposition of GMFG with the ADF-H domain recently reported in a model of ADF-H-decorated actin filament, results in a steric clash of GMFG in this region with actin. Phosphorylation of the α1-β2 loop and β5-β6 hairpin residues would change both the electrostatic surface of the region as well as the conformation of these structures and likely affect interaction with actin filaments.


    Depending on cell type and particular conditions such as cellular stress, these post-translational modifications, carried out individually or in combination, might serve to fine-tune the interaction of GMFs with actin allowing for a graded, stimulus-dependent response. Such a response could manifest itself, for example, as changes in actin filament twist. Further experiments will be required to assess the role of the GMF α1-β2 loop and determine the mechanisms regulating actin binding.
     

    Ligand Summary



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