Overview

CheY

CheY is a signal transduction protein of enteric bacteria. It mediates communication from chemotactic receptors and the flagella motor. As a default condition, the motor rotates counter-clockwise; when it interacts with the phosphorylated form of CheY, it changes to a clockwise direction. This action causes the cell to tumble, until smooth swimming is again achieved. The tumbling thus results in a change of direction. Thus, by controlling the phosphorylation state of CheY, the cell can control its swimming behavior.

The x-ray crystalographic structure of CheY from Salmonella typhimurium and Escherichi coli have been obtained (see Stock et al, 1993; Volz and Matsumura 1991; and Volz, 1993, Biochemistry 32:11741-11753). The structures are nearly identical. CheY forms a beta/alpha barrel, with secondary structure of the type:

Figure adapted from Stock et al., 1989, Microbiological Reviews 53:450-490.

The site of phosphorylation has been identified as aspartate 57. As is typical of this class of proteins, the active site is created in the crevace formed between oppositely directed loops emerging from the top of strands b-1 and b-3. Asp13 chelates a magnesium cation near the site of phosphorylation. Binding of Mg²⁺ is thought to cause the large conformational change illustrated below (the starting structure is from crystals of the unbound form (Volz and Matsumura, 1991), and the ending structure has Mg²⁺ bound (Bellsolell et al., 1994); all images in-between are fictional!; figure adapted from here).

This starting 3D image (in the left panel) shows CheY protein in the Sticks display with Structure coloring; Mg²⁺ is shown in Spacefill, colored green.

• <> View similar to that in Fig. 3 of the Stock et al. Minireview, J. Biol. Chem. 267 19753 (1992).

Lys109 is perhaps very important for function, as only lysine will suffice in this position. The only mutation that has been identified that de-regulates CheY, causing tumbling in the absence of phosphorylation, is to replace asp13 with lysine or arginine (Bourret et al., 1993, J. Biol. Chem. 268:13089-13096).

• <> Close-up view of the octahedrally coordinated Mg²⁺ site. See Stock et al., 1993. The three coordinated H₂O molecules are not shown in this starting image.
• <> Add the H₂O's (labeled "HOH").

CheA has been shown to bind to CheY. Mutations in CheY have been identified that disrupt this binding.

• <> Ball & Stick display of the residues, which when substituted, affected CheY-CheA binding. Described in Shukla & Matsumura (1995) J. Biol. Chem. 270: 24414. The CheY mutants, in increasing order of K_D, were: T87I, D13K, A90V, T112I, Y106W, E117K, E93K, V108M, and F111V.

NMR was used to probe the structure of CheY, CheY-PO4, and the asp13 mutants (Drake et al., 1993, J. Biol. Chem. 268:13081-13088; Bourret et al., 1993, J. Biol. Chem. 268:13089-13096). A small amount of the protein synthesized in vivo was labeled with F19-phenylalanine. There are 6 phenylalanine residues in the protein. The labeling procedure produced a mixture of mostly unlabled protein that also contained small quantities of protein in which one of the six residues contained F19-phe. This analogue gives an NMR signal that is very sensitive to changes in the atomic environment surrounding the labeled residue. After assigning each phe residue to its NMR signal, the signals from protein could then be monitored to determine which, if any, of the residues found themselves in a different environment after the protein was phosphorylated, or when Asp13 was mutated. Any such differences were interpreted as evidence for conformational change in the corresponding regions of the protein.

• Upon phosphorylation, the bottom of the barrel but not the top showed evidence of conformational change.
• Mutations D13K and D13R perturbed the region near K109 on the top of the barrel.
  • <> CheY (cyan cartoon), complexed with a piece of CheA (white cartoon) that binds CheY in vitro.
  • green spacefill - asp57 of CheY, the site of phosphorylation
  • orange spacefill - phenylalanines of CheY, which when fluorinated give NMR spectra suggesting altered environment upon phosphorylation
  • red spacefill - phenylalanine which when fluorinated gave NMR spectra suggesting altered environment in Asp13 mutations, which signal tumbling without phosphorylation
  • yellow spacefill - the same residues of CheY shown above, which when mutated reduce binding to the CheA fragment

These results were interpreted as evidence for a two step model of signal transduction.

• phosphorylation is the first step, perturbing the distal end of the protein,
• later steps that alter the environment of Lys109
  • the later steps were suggested to include binding of an additional magnesium cation after phosphorylation, which would alter the Lys109 environment as mimicked by D13K or D13R mutations.

Other work shows that CheY's signaling ability correlates with the conformational heterogeneity of the Tyr106 side chain.

• In the wild type, Y106 is found in both inward and outward configurations.
  • Point mutant T87I locks Y106 in an outward orientation, and the cells are smooth swimming, non-chemotactic.
  • Point mutant Y106W locks the W106 in an inward postion, and the cells tumble constantly.
  • The double mutant T87I/Y106W locks the W106 residue outward, and the cells are smooth swimming.
• These results suggest that the orientation of Y106 (or W106 in the mutant) is crucial for signalling (Zhu et al., 1997; 1996).
• In an abstract (Volz, Kalkides, Matsumuira and Dhalquist), crystal structure has been reported for chemically modified CheY in which D57 has been replaced with cysteine, and it modified to mimic phosphorylation of asparate. The structure also has Y106 locked outward, which is consistent with the above speculations.
  • The modification is cys a thiophosphonate
    (imagine adding PO₃ to the O of aspartate, and comparing that to replacing the H in -SH of cysteine with CH₂-PO₃. The resulting compounds are isosteric, but the phosphate is labile, while the thiophosphonate is stable).
    

CheB

• CheB is also a two-component signal transduction protein that functions to regulate chemotaxis in enteric bacteria.
  • <> In addition to possessing a two-component recreiver domain, CheB also has a methylesterase activity that affects chemoreceptor functions.
    • cyan cartoon - the two-component receiver domain
    • green spacefill - the site of phosphorylation
    • red, yellow and white - the methylesterase domain
    • the active site of the methylesterase is in the cleft formed by the top edge of the sheet and the packed helicees.
• The methylesterase activity is regulated by phosphorylation of the receiver domain.
• The N-terminal two-component receiver domain packs against the active site of the C-terminal domain and thus inhibits methylesterase activity by directly restricting access to the active site.

The structure suggests that phosphorylation of CheB induces a conformational change in the regulatory domain that disrupts the domain interface, resulting in a repositioning of the domains and allowing access to the active site (Djordjevic et al).

NarL

<> NarL is a two component response protein whose receiver domain (red - a helicees; yellow - b-strands; green - asp54 site of phosphorylation) controls a DNA binding output function (cyan). (Baikalov et al, 1996)

• It was the first two component receiver domain that is covalently attached to an output domain (as most are) for which structure is available.
  • Note that in this structure of the unphosphorylated protein, the linker region is not seen in the diffraction data, presumably indicating that it is mobile.
• As in CheY, in NarL the two component receiver domain, the strands (yellow) form the core sheet around which the helicees (red) pack.
• The site of phosphorylation is asp54 (green).
  • no structure information is available for the activated form of NarL
• The DNA binding domain is prevented from binding to DNA in the inactive form of the protein.

The working hypothesis is that phosphorylation releases the DNA binding surface so that it can contact the DNA (Baikalov et al, 1998)

• Note that a different part of the two-component receiver domain packs against the DNA binding domain in NarL than packs against the methylesterase domain of CheB.
• This indicates that signal transduction can be structurally linked to different parts of the two-componet receiver module.

NtrC

NTRC is a transcriptional enhancer binding protein whose N-terminal domain is a member of the family of receiver domains of two-component regulatory systems.  

• It is phosphorylated on aspartate 54, by NtrB, and organic phosphates (see Feng et al, 1992).
• <> The solution structure of the N-terminal receiver domain of the NTRC protein was determined by NMR. (Volkman et al, 1995)
  • yellow - b strands
  • red - alpha helicees
  • white - coils
• Comparison of this solution structure with the crystal structures of the homologous protein CheY in both the Mg(2+)-free and Mg(2+)-bound forms reveals a very similar fold, with the only significant difference occurring in the positioning of helix 4 relative to the rest of the protein.
• Examination of the conformation of consensus residues of the receiver domain superfamily [Volz, K. (1993) Biochemistry 32, 11741-11753] in the structures of the NTRC receiver domain and CheY
  • establishes the structural importance of residues whose side chains are involved in hydrogen bonding or hydrophobic core interactions.
  • and reveals that some nonconsensus residues may be conserved for their ability to fulfill helix capping roles

Combinations of point mutations resulted in both increased structural changes in the N-terminal domain, monitored by NMR chemical shift differences, and increased transcriptional activation by the full-length protein. (Nohaile et al., 1997)

• <> Structural changes caused by substitutions outside the active site (D86N and A89T) were not only local but extended over a substantial portion of the N-terminal domain including the region from alpha-helix 3 to beta-strand 5 ("3445 face") and propagating to the active site.
• The activating substitution of glutamate for aspartate at the site of phosphorylation (D54E) also triggered structural changes in the 3445 face.

These results were interpreted as evidence for an interaction between the site of phosphorylation and the 3445 face, which somehow leads to signal transduction upon phosphorylation.

• There is overlap but not identity between this predicted surface of NtrC's receiver domain and that which packs against the methylesterase in CheB
  • NtrC: helix-3, strand-4, helix-4, strand-5
  • CheB: helix-4, strand-5, and helix-5

Heterotrimeric G-Proteins of Eukaryotes

Examples of the two-component signal transduction system are known in fungal and plant eukaryotes. In other eukaryotes there is no evidence of two-component signal transduction; however, a signal transduction system known as the heterotrimeric G-proteins that is found in these organisms bears some very strong similarities to the two-component signal transduction paradigm.

• Each heterotrimeric G protein consists of three different subunits:
  • alpha,
  • beta, and
  • gamma.
• The overall fold of the alpha subunit is very similar to that of the two-component receiver module.
• In the same cleft near the top of the b-sheet, the alpha subunit has a site for binding GDP or GTP and a catalytic center for hydrolyzing GTP to GDP.
• The role of GTP binding, catalysis and release is in many ways analogous to the phosphorylation and de-phosphorylation of two-component receiver domains.
• Usually, both the alpha subunit and the gamma subunit have lipid anchors to permit lateral diffusion, protein-lipid, and protein-protein interactions
  • alpha subunit - fatty acyl lipid anchor.
  • gamma subunit - isoprenoid lipid anchor.

Rather than recreating structural aids, I point you to a set prepared by George M. Helmkamp, Jr., Ph.D. as part of a course in biochemistry at Kansas University Medical Center (KUMC). This course has a collection of really great learning aids. In addition to Rasmol Chime scripts (for which you need the pluggin Chime), Dr. Helmkamp also makes use of Macromedia's Shockwave pluggin for animations in some of the other lectures.

Important reminder

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  • launch a separate instance of your browser:
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