Metal-Mediated Affinity and Orientation Specificity in a Computationally Designed Protein Homodimer

Computationally designing protein–protein interactions with high affinity and desired orientation is a challenging task. Incorporating metal-binding sites at the target interface may be one approach for increasing affinity and specifying the binding mode, thereby improving robustness of designed interactions for use as tools in basic research as well as in applications from biotechnology to medicine. Here we describe a Rosetta-based approach for the rational design of a protein monomer to form a zinc-mediated, symmetric homodimer. Our metal interface design, named MID1 (NESG target ID OR37), forms a tight dimer in the presence of zinc (MID1-zinc) with a dissociation constant <30 nM. Without zinc the dissociation constant is 4 μM. The crystal structure of MID1-zinc shows good overall agreement with the computational model, but only three out of four designed histidines coordinate zinc. However, a histidine-to-glutamate point mutation resulted in four-coordination of zinc, and the resulting metal binding site and dimer orientation closely matches the computational model (Cα rmsd = 1.4 Å)

Crystal Structure of the Munc13–1 C ₂A Domain Homodimer

<div><p>(A) A region of the 2F₀-F_c electron density contoured at the 1σ level. </p> <p>(B) Ribbon diagram of the Munc13–1 C₂A domain homodimer formed by monomers A (blue) and C (orange) showing a top view of the β-barrel like structure. The β-strands are labeled with numbers, and the N- and C-termini are indicated with N and C, respectively. </p> <p>(C) Superposition of monomers A and C.</p> <p>(D) Superposition of monomer C of the Munc13–1 C₂A domain (orange) and the C₂ domain of PLC-δ1 (green). </p> <p>(E) Surface representation of the Munc13–1 C₂A-domain homodimer. </p> <p>(F) Ribbon diagram of the Munc13–1 C₂A-domain homodimer showing a view perpendicular to that of (B) and illustrating the intermolecular strand–strand interactions that close the β-barrel. The backbone atoms from strand 3 of monomer A and strand 6 of monomer C involved in strand–strand hydrogen bonds are shown as stick models. </p> <p>(G) Close-up view of the dimerization interface. The side chains from residues involved in intermolecular contacts and the Cα carbons of the same residues are shown as stick models, with oxygen atoms in red and nitrogen atoms in blue; Cα carbons are shown with the same color as the ribbon, and other carbons are shown in gray for monomer A and yellow for monomer C. The carbonyl groups of I70, which form hydrogen bonds (dotted lines) with the S33 hydroxyl groups are also shown as stick models. For simplicity, other hydrogen bonds are not shown.</p> <p>All diagrams were generated with Pymol (DeLano Scientific, San Carlos, California).</p></div

The Munc13–1 C ₂A Domain Homodimerizes

<div><p>(A) Domain structure of Munc13–1 and RIM2α. The calmodulin-binding sequence (CaMb) of Munc13–1 and the helices that flank the RIM2α ZF domain (labeled a1 and a2) are indicated below the diagrams, and residue numbers are indicated above them.</p> <p>(B) Gel filtration analysis of Munc13–1_3–150 (black), RIM2α_82–142 (blue), and the complex between them (red). Elution volumes of two molecular standards are indicated at the top. </p> <p>(C)¹H-¹⁵N HSQC spectrum of¹⁵N-labeled Munc13–1_3–150 at 500 MHz. </p> <p>(D)¹H-¹⁵N HSQC spectrum of¹⁵N-labeled Munc13–1_3–150 bound to unlabeled RIM2α_82–142 at 500 MHz. </p> <p>(E) Equilibrium sedimentation analysis of Munc13–1_3–128. The data were obtained at centrifugation speeds of 20,000 rpm (black), 25,000 rpm (red), 30,000 rpm (green), and 35,000 rpm (blue). Curves in the bottom panel were generated by fitting the data to a monomer-dimer equilibrium model. The top panel shows the residuals. </p> <p>mAU, milliabsorbance units; ppm, parts per million.</p></div

Interplay between the Interactions of Munc13–1, α-RIM, and Rab3s

<div><p>(A) Structures of the Munc13–1 C₂A-domain homodimer and the Munc13–1_3–150(K32E)/RIM2α_82–142 heterodimer superimposed using monomer C of the homodimer (green) and monomer A of the heterodimer (orange). Ribbon diagrams and transparent surface representations are shown for monomer A of the homodimer (red) and RIM2α_82–142 (blue) to illustrate the steric clash that would occur if both complexes co-existed. The K99 side chain of the ZF domain is shown as a space-filling model. The inset shows a close-up of the region where this side chain (shown here as stick model) would insert into the homodimer interface (the surface representations are not shown). </p> <p>(B) Structures of the Munc13–1_3–150(K32E)/RIM2α_82–142 heterodimer and the rabphilin/Rab3A complex [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040192#pbio-0040192-b027" target="_blank">27</a>] superimposed using the ZF domains of RIM2α (blue) and rabphilin (green). Ribbon diagrams and transparent surface representations are shown for Munc13–1_3–150(K32E) (orange) and Rab3A (purple). Zinc atoms are shown as yellow (RIM2α) or red (rabphilin) spheres. The SGAWFF motif of rabphilin is shown as cyan stick model. </p> <p>(C) Model of a cascade of protein–protein interactions that may regulate synaptic vesicle priming and presynaptic plasticity. Munc13–1 is shown in orange and Rab3 in purple. α-RIM is shown in blue (ZF domain), green (helices a1 and a2), and red (SGAWFY motif). The model represents how formation of the tripartite Munc13–1/α-RIM/Rab3 complex involves dissociation of the Munc13–1 homodimer and release of the interaction between the SGAWFY motif and Rab3A.</p></div

Evolutionary Conservation of Residues Involved in Munc13–1 Homodimerization and Munc13–1/α-RIM Heterodimerization

<p>(A) and ( B) Sequence alignments of the Munc13/Unc13 N-terminal sequences including the C₂A domain and the C-terminal α-helical extension (C-ter) (A), and of the α-RIM/Unc10 ZF domains (B). The eight cysteine residues conserved in all α-RIM/Unc10 ZF domains are shown in white on a black background. Residues conserved in more than 70% of the sequences are highlighted in white with a red background. Residues involved in homo and heterodimerization are indicated by an asterisk (*) above the sequences. The secondary structure elements are indicated below the alignments. Species abbreviations: Am, <i>Apis mellifera;</i> Ce, <i>Caenorhabditis elegans;</i> Dm, <i>Drosophila melanogaster;</i> Dn, <i>Danio rerio;</i> Rn, <i>Rattus norvegicus;</i> Tn, <i>Tetraodon nigroviridis;</i> Xt, <i>Xenopus tropicalis.</i></p

Crystal Structure of the Munc13–1 _3–150(K32E)/RIM2α ZF Domain Complex

<div><p>(A) A region of the 2F₀-F_c electron density contoured at the 1σ level. </p> <p>(B) Ribbon diagram of the complex with 2:1 stoichiometry seen in the crystals, with RIM2α_82–142 shown in blue and monomers A and B of Munc13–1_3–150(K32E) shown in orange and green, respectively. Zinc atoms are shown as yellow spheres. The N- and C-termini are indicated with N and C, respectively. </p> <p>(C) Superposition of monomers A and B of Munc13–1_3–150(K32E) observed in the crystals. The β-strands are labeled with numbers. </p> <p>(D) Superposition of monomer A of Munc13–1_3–150(K32E) (orange) with monomer C of the Munc13–1 C₂A-domain homodimer (green). </p> <p>(E) Superposition of the structure of the RIM2α ZF domain observed in the heterodimer (blue) and its solution structure determined in isolation by NMR spectroscopy (red) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040192#pbio-0040192-b020" target="_blank">20</a>]. Only the zinc atoms from the former structure are shown (yellow spheres). The β-strands are labeled with numbers, and the C-terminal α-helix is labeled a2. </p></div

The K32A Mutation Preserves Munc13–1/RIM2α ZF Domain Heterodimerization

<div><p>(A) Gel filtration analysis of Munc13–1_3–150(K32E) (black), RIM2α_82–142 (blue), and the complex between them (red). </p> <p>(B) ITC analysis of the binding of Munc13–1_3–150(K32E) to RIM2α_82–142. </p> <p>(C)¹H-¹⁵N HSQC spectra of¹⁵N-labeled Munc13–1_3–150(K32E) alone (black) and bound to unlabeled RIM2α_82–142 (red) at 800 MHz. </p> <p>(D)¹H-¹⁵N HSQC spectra of the¹⁵N-Munc13–1_3–150(K32E)/RIM2α_82–142 complex (red) and the wild-type¹⁵N-Munc13–1_3–150/RIM2α_82–142 complex (black) at 800 MHz. Note that the different appearance of the latter spectrum from that shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040192#pbio-0040192-g001" target="_blank">Figure 1</a>D arises from the different magnetic field used. New well-resolved cross-peaks that appear at the edges of the spectrum upon heterodimer formation are circled in blue in (C) and (D). </p> <p>mAU, milliabsorbance units; ppm, parts per million.</p></div

Heterotrimeric G-protein Signaling Is Critical to Pathogenic Processes in <em>Entamoeba histolytica</em>

<div><p>Heterotrimeric G-protein signaling pathways are vital components of physiology, and many are amenable to pharmacologic manipulation. Here, we identify functional heterotrimeric G-protein subunits in <em>Entamoeba histolytica</em>, the causative agent of amoebic colitis. The <em>E. histolytica</em> Gα subunit EhGα1 exhibits conventional nucleotide cycling properties and is seen to interact with EhGβγ dimers and a candidate effector, EhRGS-RhoGEF, in typical, nucleotide-state-selective fashions. In contrast, a crystal structure of EhGα1 highlights unique features and classification outside of conventional mammalian Gα subfamilies. <em>E. histolytica</em> trophozoites overexpressing wildtype EhGα1 in an inducible manner exhibit an enhanced ability to kill host cells that may be wholly or partially due to enhanced host cell attachment. EhGα1-overexpressing trophozoites also display enhanced transmigration across a Matrigel barrier, an effect that may result from altered baseline migration. Inducible expression of a dominant negative EhGα1 variant engenders the converse phenotypes. Transcriptomic studies reveal that modulation of pathogenesis-related trophozoite behaviors by perturbed heterotrimeric G-protein expression includes transcriptional regulation of virulence factors and altered trafficking of cysteine proteases. Collectively, our studies suggest that <em>E. histolytica</em> possesses a divergent heterotrimeric G-protein signaling axis that modulates key aspects of cellular processes related to the pathogenesis of this infectious organism.</p> </div

EhGα1 cycles between an active, GTP-bound state and an inactive, GDP-bound state.

<p>(<b>A</b>) EhGα1 bound non-hydrolyzable GTPγS as determined by radionucleotide binding. The observed exchange rate, <i>k_obs</i> = 0.27 min⁻¹ ±0.06, indicates faster spontaneous GDP release than human Gα_i1 (<i>k_obs</i> = 0.06 min⁻¹ ±0.01). (<b>B</b>) EhGα1 hydrolyzed GTP[γ-³²P] at 0.21 min⁻¹ ±0.02, as determined by single turnover hydrolysis assays. No difference was observed for selenomethionine, lysine-methylated EhGα1 used for crystallization. (<b>C</b>) EhGα1 changes conformation upon binding the transition state mimetic aluminum tetrafluoride. Intrinsic EhGα1 fluorescence following excitation at 285 nm increases upon activation, reflecting burial of a conserved tryptophan on switch 2 (Trp-196). (<b>D</b>) EhGα1 adopts an active switch conformation upon addition of the non-hydrolyzable GTP analog GppNHp, as reflected by increased intrinsic tryptophan fluorescence. The kinetics of GppNHp-mediated activation are consistent with the kinetics of radiolabeled GTP analog binding (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003040#ppat-1003040-g001" target="_blank">Fig. 1A</a>). In contrast, addition of hydrolyzable GTP does not result in EhGα1 activation, indicating that nucleotide exchange, rather than GTP hydrolysis, is the rate-limiting step in the nucleotide cycle of EhGα1. (<b>E, F</b>) Two EhGα1 point mutants were profiled for effects on nucleotide cycle. The dominant negative S37C possessed negligible GTP binding. The constitutively active Q189L bound but did not hydrolyze GTP. Error bars in all panels represent standard error of the mean.</p

Evolutionary relationship of Gα subunits and identification of EhRGS-RhoGEF as a putative effector for activated EhGα1.

<p>(<b>A</b>) Gα subunit protein sequences from <i>E. histolytica</i>, <i>D. discoideum</i> (D.d.), <i>A. thaliana</i> (A.t.), <i>S. cerevisiae</i> (S.c.), <i>D. melanogaster</i> (D.m), and <i>H. sapiens</i> (H.s.) were aligned and a bootstrapping consensus phylogram created using MEGA5 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003040#ppat.1003040-Tamura1" target="_blank">[41]</a>. Bootstrap values are indicated at each branch point. EhGα1 is distantly related to metazoan Gα subunits, specifically the adenylyl cyclase stimulatory Gα_s, adenylyl cyclase inhibitory Gα_i/o, phospholipase Cβ coupled Gα_q, and RGS-RhoGEF activating Gα_12/13 subfamilies. (<b>B</b>) Recombinant EhRGS-RhoGEF protein was immobilized on a surface plasmon resonance chip and EhGα1 protein flowed over in one of two nucleotide states. The EhRGS-RhoGEF biosensor bound EhGα1 selectively in the activated, GDP·AlF₄⁻-bound state (AMF).</p