1. Modulation of Host Signaling by a Bacterial Mimic
C.Erec Stebbins, Jorge E Galán 
Molecular Cell 
Volume 6, Issue 6, Pages (December 2000)
DOI: /S (00)

2. Figure 1
Proteolytic Digestion of the Salmonella typhimurium Effector SptP Delineates Functional GAP and Tyrosine Phosphatase Domains
(A) Schematic summarizing the domain organization and the points of proteolytic digestion of SptP (arrow labeled 161). Regions of homology to related virulence factors are indicated by shading, the translocation domain is shown in white, and the fragment crystallized is denoted by a thick line with arrows. The key catalytic residues Arg-209 and Cys-481 are labeled.
(B) SptP161–543 retains GAP activity toward Rac1. Purified SptP161–543 was added to purified [γ-32P]GTP-loaded GST-Rac1 (400 nM) and the GAP activity measured by a filter binding assay (Self and Hall 1995).
(C) SptP161–543 possesses tyrosine phosphatase activity. The tyrosine phosphatase activity was measured using a tyrosine phosphorylated peptide derived from human hirudim in the presence and absence of the tyrosine phosphatase inhibitors sodium vanadate and sodium tungstate (Kaniga et al. 1996).
(D) SptP161–543 and Rac1 form a stable and long-lived complex in the presence of aluminum fluoride, as assayed by gel filtration and visualized by SDS–PAGE stained with Coomassie blue. Superimposed over the protein bands is the chromatography absorption profile.
Molecular Cell 2000 6, DOI: ( /S (00) )

3. Figure 2
Tertiary Structure of SptP–Rac1 Heterodimer and Primary Sequence Alignments of Related Homologs
(A) The AlF3 transition state complex reveals that SptP consists of two domains: a COOH-terminal tyrosine phosphatase fold (violet coloring in the diagram) and an NH2-terminal four-helix bundle GAP domain (blue) that inserts a catalytic arginine (shown in white with nitrogens colored blue) into the active site of the GTPase Rac1 (shown in green with the bound GDP nucleotide). The catalytic cysteine of the phosphatase domain (with yellow sulfur) is also illustrated. The key regulatory and enzymatic elements are also shown: for the GTPase, Switch I (orange) and Switch II (red); for the phosphatase, the P loop (orange) and the WPD loop (green). The secondary structural elements for the GAP domain are also labeled. N, NH2 terminus; C, COOH terminus.
(B) Structure-based sequence alignments of related bacterial virulence factors with the SptP GAP domain generated by sequence threading. Helices maintaining the four-helix bundle architecture are colored blue. Absolutely conserved residues are shown in yellow, while those that contact the Switch I, Switch II, and GDP elements of Rac1 are shown in blue, green, and orange, respectively. Residues participating in the intramolecular interface with the phosphatase domain are shown in purple. The catalytic arginine is shown in red. Residues that are part of the GAP domain hydrophobic core are underscored by asterisks, and the hydrophobic positions of the heptad repeats (“a” and “d”) are labeled. The solvent accessibility is indicated by a shaded box over each amino acid.
(C) Structure-based sequence alignments of the key regulatory elements of related tyrosine phosphatase domains with SptP and homologous GTPases with Rac1. Absolutely conserved residues are shown in yellow, and catalytic residues are shown in red. Residues in Rac1 involved in contacting SptP are shown in blue. The solvent accessibility is indicated by a shaded box over each amino acid.
Molecular Cell 2000 6, DOI: ( /S (00) )

4. Figure 3
The Tyrosine Phosphatase Domain of SptP Harbors a Conserved Active Site but Distinct Surface Properties
(A) Structural alignment of the SptP phophatase domain with the PTP1B and YopH homologs.
(B) Key residues in the active site of the phosphatase domain are positioned similarly to those in related enzymes and coordinate solvent and substrate mimics analogously. Main chain coloring is as in Figure 2, and side chains are shown in purple. Highly ordered water molecules are shown as magenta spheres. Hydrogen bonds are shown as white dotted lines, yellow dotted lines when involving Cys-481, or as solid blue lines for particularly strong bonds with distances less than 2.5 Å. Tungstate from the refined structure of a heavy atom soak is superimposed on the Apo structure. (C) Illustration of sequence divergence between SptP and YopH as mapped to the molecular surface of SptP. Sequence aligned residues are colored on a gradient from absolute conservation (blue) to divergent (red), with the portion of the SptP molecular surface corresponding to a given residue adopting its color. (D) Comparison of the aligned phosphatase domains of SptP and YopH illustrated with a molecular surface colored by electrostatic potential such that red is negative (acidic) and blue is positive (basic). “Front” and “back” views are given, which are related by the 180° rotation about the axis indicated.
Molecular Cell 2000 6, DOI: ( /S (00) )

5. Figure 4
The Interface with Rac1 Is Extensive and Highly Complementary to the Switch I, Switch II, and Nucleotide Regions of the GTPase
(A) The extensive surface charge complementarity between the GAP domain and Rac1 is illustrated with a molecular surface colored by electrostatic potential such that red is negative (acidic) and blue is positive (basic). Arrows indicate regions of charge complementarity between the Rac1 and SptP surfaces. (B and C) The SptP GAP domain (secondary structure shown in blue and side chains in cyan) interact with the Switch I (yellow) and Switch II (red) regulatory elements of Rac1 (with yellow side chains). Hydrogen bonds are indicated by white dotted lines, and the atoms of nitrogen and oxygen are show in blue and red, respectively. Water molecules are shown as large magenta spheres. A large “W” indicates the nucleophilic water molecule positioned by Gln-61 of Rac. (D) SptP positions Gln-61 of Rac1 through molecular contacts and inserts Arg-209 into the active site to stabilize the transition state. Hydrogen bonds are indicated by white dotted lines or as smaller gray dotted lines for weak bonds. AlF3 is shown with the fluorides colored brown and the aluminum gray. The magnesium ion and water molecules are shown as large blue or magenta spheres, respectively. GDP carbon bonds are shown in yellow. A large “W” indicates the nucleophilic water molecule positioned by Gln-61 of Rac1. The phosphate binding (P loop) and guanine binding loops (G loops) of Rac1 are shown in purple. The bonds proposed to form during the phosphoryl transfer are shown as solid white lines. (E) The interface between the GAP (blue) and tyrosine phosphatase (purple) domains of SptP consists of several direct and water-mediated hydrogen bonds as well as a small hydrophobic interface.
Molecular Cell 2000 6, DOI: ( /S (00) )

6. Figure 5
The GAP Domain of SptP Undergoes Marked Structural Changes upon Binding Rac1
A comparison of the atomic B factors between the SptP monomer and the SptP–Rac1 heterodimeric transition state complex is shown. The ribbon diagrams are colored according to an absolute gradient in the B factor from blue (ordered with a low B factor) through red (disordered with a high B factor). Connectivity missing due to disorder in the monomer is represented by black dotted lines based on their conformation in the heterodimer.
Molecular Cell 2000 6, DOI: ( /S (00) )

7. Figure 6
Structure-Based Mutagenesis Reveals Impairment of In Vitro and In Vivo Activity of SptP
(A) In vitro analysis of complex formation of SptP mutants by gel filtration chromatography. Excess Rac1 (see load lane) was added to bias the assay toward formation of the SptP–Rac1 complex. The chromatograph for each sample is shown in different colors and the corresponding SDS–PAGE analysis of the elution fractions is shown underneath. The wild-type and G252A mutants show the presence of the SptP–Rac1 heterodimer in the 14 and 15 ml fractions (corresponding to a molecular weight of 62 kDa) and little or no evidence for the monomeric SptP species (42 kDa). The mutants T213A, Q246A, and T249A lack any evidence for the heterodimeric species, show a strong signal for monomeric SptP, and an increased signal for the Rac1 monomer. (B) Enzymatic analysis of the SptP mutants by a filter binding assay. After 10 min, the wild-type and G252A mutants have hydrolyzed most of the Rac1-bound GTP, whereas the mutants that are impaired in SptP–Rac1 complex formation, T213A, Q246A, and T249A, possess little if no specific GAP activity. (C) The catalytically impaired SptP mutants (T213A, Q246A, and T249A) also failed to complement a S. typhimurium sptP null mutant in an actin cytoskeleton recovery assay. In contrast, the biochemically active G252A mutant complemented the sptP null mutant, leading to wild type levels of cellular recovery after infection.
Molecular Cell 2000 6, DOI: ( /S (00) )

8. Figure 7
Comparisons of GAP Enzymes and Their Interactions with Rho GTPases
(A) Comparison of the SptP GAP domain and host GAP enzymes reveal that they engage the GTPase in a similar manner, but SptP is differentiated by its smaller size and different fold. The GTPases are shown as molecular surfaces colored gray, except for the Switch I (orange) and Switch II (red) regulatory domains and the nucleotide GDP (green). (B) Alignment of Cdc42 and RhoA from their respective GAP complexes (with Cdc42GAP and RhoGAP) to Rac1 from the complex with SptP. Switch I of Rac1 is shown in orange. The RhoA and Cdc42 main chain traces are shown in gray. Residues with significant positional variation between the Rho GTPase–GAP transition state complexes are shown in orange, purple, or yellow for Rac1, Cdc42, and RhoA, respectively, while the residues in SptP which they contact are cyan. Pro-34 (Rac1 numbering) of RhoA perfectly superimposes with Pro-34 of Cdc42, and the yellow of this residue is not visible.
Molecular Cell 2000 6, DOI: ( /S (00) )