The structural basis of Arf effector specificity: the crystal structure of ARF6 in a complex with JIP4.

The JNK-interacting proteins, JIP3 and JIP4, are specific effectors of the small GTP-binding protein ARF6. The interaction of ARF6-GTP with the second leucine zipper (LZII) domains of JIP3/JIP4 regulates the binding of JIPs to kinesin-1 and dynactin. Here, we report the crystal structure of ARF6-GTP bound to the JIP4-LZII at 1.9 A resolution. The complex is a heterotetramer with dyad symmetry arranged in an ARF6-(JIP4)(2)-ARF6 configuration. Comparison of the ARF6-JIP4 interface with the equivalent region of ARF1 shows the structural basis of JIP4's specificity for ARF6. Using site-directed mutagenesis and surface plasmon resonance, we further show that non-conserved residues at the switch region borders are the key structural determinants of JIP4 specificity. A structure-derived model of the association of the ARF6-JIP3/JIP4 complex with membranes shows that the JIP4-LZII coiled-coil should lie along the membrane to prevent steric hindrances, resulting in only one ARF6 molecule bound. Such a heterotrimeric complex gives insights to better understand the ARF6-mediated motor switch regulatory function.

Oligomerization of the SPP1 scaffolding protein.

Viral scaffolding proteins direct polymerization of major capsid protein subunits into icosahedral procapsid structures. The scaffolding protein of bacteriophage SPP1 was engineered with a C-terminal hexahistidine tag (gp11-His(6)) and purified. The protein is an alpha-helical-rich molecule with a very elongated shape as found for internal scaffolding proteins from other phages. It is a 3.3 S tetramer of 93.6 kDa at micromolar concentrations. Intersubunit cross-linking of these tetramers generated preferentially covalently bound dimers, revealing that gp11-His(6) is structurally a dimer of dimers. Incubation at temperatures above 37 degrees C correlated with a reduction of its alpha-helical content and a less effective intersubunit cross-linking. Complete loss of secondary structure was observed at temperatures above 60 degrees C. Refolding of gp11-His(6) thermally denatured at 65 degrees C led to reacquisition of the protein native ellipticity spectrum but the resulting population of molecules was heterogeneous. Its hydrodynamic behavior was compatible with a mix of 3.3 S elongated tetramers (approximately 90%) and a smaller fraction of 2.4 S dimers (approximately 10%). This population of gp11-His(6) was competent to direct polymerization of the SPP1 major capsid protein gp13 into procapsid-like structures in a newly developed assembly assay in vitro. Although native tetramers were active in assembly, refolded gp11-His(6) showed enhanced binding to gp13 revealing a more active species for interaction with the major capsid protein than native gp11-His(6).

Archease from Pyrococcus abyssi improves substrate specificity and solubility of a tRNA m5C methyltransferase.

Members of the archease superfamily of proteins are represented in all three domains of life. Archease genes are generally located adjacent to genes encoding proteins involved in DNA or RNA processing. Archease have therefore been predicted to play a modulator or chaperone role in selected steps of DNA or RNA metabolism, although the roles of archeases remain to be established experimentally. Here we report the function of one of these archeases from the hyperthermophile Pyrococcus abyssi. The corresponding gene (PAB1946) is located in a bicistronic operon immediately upstream from a second open reading frame (PAB1947), which is shown here to encode a tRNA m(5)C methyltransferase. In vitro, the purified recombinant methyltransferase catalyzes m(5)C formation at several cytosines within tRNAs with preference for C49. The specificity of the methyltransferase is increased by the archease. In solution, the archease exists as a monomer, trimer, and hexamer. Only the oligomeric states bind the methyltransferase and prevent its aggregation, in addition to hindering dimerization of the methyltransferase-tRNA complex. This P. abyssi system possibly reflects the general function of archeases in preventing protein aggregation and modulating the function of their accompanying proteins.

Structural basis for ARF1-mediated recruitment of ARHGAP21 to Golgi membranes.

ARHGAP21 is a Rho family GTPase-activating protein (RhoGAP) that controls the Arp2/3 complex and F-actin dynamics at the Golgi complex by regulating the activity of the small GTPase Cdc42. ARHGAP21 is recruited to the Golgi by binding to another small GTPase, ARF1. Here, we present the crystal structure of the activated GTP-bound form of ARF1 in a complex with the Arf-binding domain (ArfBD) of ARHGAP21 at 2.1 A resolution. We show that ArfBD comprises a PH domain adjoining a C-terminal alpha helix, and that ARF1 interacts with both of these structural motifs through its switch regions and triggers structural rearrangement of the PH domain. We used site-directed mutagenesis to confirm that both the PH domain and the helical motif are essential for the binding of ArfBD to ARF1 and for its recruitment to the Golgi. Our data demonstrate that two well-known small GTPase-binding motifs, the PH domain and the alpha helical motif, can combine to create a novel mode of binding to Arfs.

PhLP3 modulates CCT-mediated actin and tubulin folding via ternary complexes with substrates.

Many ATP-dependent molecular chaperones, including Hsp70, Hsp90, and the chaperonins GroEL/Hsp60, require cofactor proteins to regulate their ATPase activities and thus folding functions in vivo. One conspicuous exception has been the eukaryotic chaperonin CCT, for which no regulator of its ATPase activity, other than non-native substrate proteins, is known. We identify the evolutionarily conserved PhLP3 (phosducin-like protein 3) as a modulator of CCT function in vitro and in vivo. PhLP3 binds CCT, spanning the cylindrical chaperonin cavity and contacting at least two subunits. When present in a ternary complex with CCT and an actin or tubulin substrate, PhLP3 significantly diminishes the chaperonin ATPase activity, and accordingly, excess PhLP3 perturbs actin or tubulin folding in vitro. Most interestingly, however, the Saccharomyces cerevisiae PhLP3 homologue is required for proper actin and tubulin function. This cellular role of PhLP3 is most apparent in a strain that also lacks prefoldin, a chaperone that facilitates CCT-mediated actin and tubulin folding. We propose that the antagonistic actions of PhLP3 and prefoldin serve to modulate CCT activity and play a key role in establishing a functional cytoskeleton in vivo.

Modulating fibroblast cell proliferation with functionalized poly(methyl methacrylate) based copolymers: chemical composition and monomer distribution effect.

Poly(methyl methacrylate)-based terpolymers bearing sulfonate and carboxylate groups have been synthesized by radical copolymerization leading to polymers with random distributions of ionic monomer units. Fibroblast cells were seeded on terpolymers of various molar compositions of ionic groups. Kinetics of the cell proliferation were examined and systematically compared to the nonfunctionalized control polymer, poly(methyl methacrylate). Modulation of cell proliferation was observed on 15% ionic monomer content copolymers of various compositions (R = COO(-)/(COO(-) + SO(3)(-)) and varies from 0 to 1). The inhibition percentage of cell proliferation calculated for each polymer by comparison to the cell proliferation on the control was plotted against R and gave a maximum value for R close to 0.55. Copolymers with ionic group contents higher or lower than 15% exhibit inhibition percentages fitting with those previously observed for the same R values, showing that the hydrophilic properties are not sufficient to explain the modulation effect of this material toward cells. Moreover, for each polymer tested, cells, even if inhibited in growth, were shown to be viable, indicating that the synthesized terpolymers exhibit cytostatic properties excluding any cytotoxic effect. Such polymers may be used for the fabrication of biocompatible intraocular lenses and prevent secondary cataract.