The N-terminal Receiver Domain of
Nitrogen Regulatory Protein C

Carla Carroll and David Marcey
CLU Biology Department
©David Marcey, 2001, 2003

I. Introduction
II. The active site in the receiver domain
III. Conformation changes induced by phosphorylation
IV. Implications
V. Web Resources on NtrC and nitrogen metabolism
VI. References

Note:  This exhibit is best viewed if the cue buttons ( ) are pressed in sequence and if the viewer does not independently manipulate the molecule on the left.

This exhibit, based on the recent work of Kern, et al. (1999), explores the Nitrogen Regulatory Protein C (NtrC). NtrC is a positively acting bacterial transcription factor that is involved in regulating the metabolism of nitrogen. The structure shown at left is the N-terminal receiver domain of NtrC, which makes up one unit of a two component regulatory system. The other unit is an upstream sensor kinase that transfers a phosophate to NtrC when changes in nitrogen concentration are detected. The receiver domain of NtrC is thus activated by phosphorylation and sends a signal to the NtrC transcriptional activator domain via structural rearrangements. This causes NtrC to oligomerize, to hydrolyze ATP, and to activate transcription of particular response genes that encode proteins involved in nitrogen metabolism (e.g. Volkman et al, (1995). Transcription of these genes is activated through NtrC interaction with the sigma 54-holoenzyme form of RNA polymerase. Sigma 54, or NtrA, is an alternative sigma subunit of RNA polymerase that allows the polymerase to recognize a novel class of promoters.

The N-terminal receiver domain is homologous to a large family of signal transduction receiver domains (see ref. 5). This superfamily has highly conserved active site residues that are located in the first beta strand . Two aspartate residues (D10 and D11) are usually followed by an additional asp (D12) or a glutamate.

The receiver domain contains an alpha/beta twisted open-sheet structure, comprising two beta-alpha-beta motifs that are connected by an alpha helix . Two adjacent beta strands form a binding crevice lined by two adjacent loop regions . Positioned in the crevice is the active site aspartate (D54) where phosphorylation occurs , as well as the previously mentioned asps (D10, D11, and D12.

III. Conformation changes induced by phosphorylation of the active site

Although NtrC is not the first receiver domain to be studied, it is the first that has been studied in its active (phosphorylated) state. Low stability of the phosphoaspartate linkage has hindered researchers in the past from studying activated receiver domains.

NtrC is activated upon phosphorylation of the active site asp, catalyzed by histidine kinase. This action requires ATP. Removing the receiver domain does not substitute for phosphorylation, which implies that a new interaction between domains must occur after phosphorylation (Kern, et al., 1999).

Shown at left are the structures of unphosphorylated (top) and phosphorylated (bottom) NtrC receiver domains. Examination of these illustrates several key structural changes that occur upon phosphorylation of the active site:

• The change of the position of a Mg⁺⁺ would lead to an electrostatic repulsion between the phosphate group attached to the active site asp and the side chains of: asp10, asp11, and possibly asp12. This repulsion may act in separating the active site from these aspartates.
• The backbone at arg56 would then be released , enabling the rearrangement of the loop above the active site asp that connects beta strand 3 to alpha-helix 3. This rearrangement allows the reorganization of the '3445' face of the molecule (alpha helix 3, alpha helix 4, beta strand 4,and beta strand 5) which tilts to the left away from the active site. Structural rearrangement of the '3445' face upon phosphorylation of asp54 suggests that the active site and the '3445' face interact (see ref.5).
• Phosphorylation-induced conformation changes provide new interactions between residues ala89 and met81 and between asp93 and ile80 and tyr10.

• A pronounced rearrangement of the loops leading into and out of alpha-helix 4 as well as axial rotation of this helix by ~100^o occurs as a result of phosphorylation. This axial rotation causes key hydrophobic side chains of alpha-helix 4 (leu87, ala90, and val91) to rotate from the core to the outside of the receiver domain. Rotation thus exposes a new hydrophobic surface for potential interaction with the NtrC activation domain. This may be seen by examining the electrostatic potential of the surface of helix 4 (allow time to load surface view). In this view, red, white, and blue indicate positive, neutral, and negative surface potentials, respectively.

All of these rearrangements are likely to be important for binding of NtrC to the downstream target, the NtrC activation domain.

IV. Implications

Two component signal transduction pathways are commonly found in many eubacterial responses to environmental cues, including nitrogen regulation (NtrC), chemotaxis (CheY), and osmoregulation. NtrC and CheY belong to a family of receiver domains that has highly conserved active site residues and tertiary structure. Study of NtrC's structural changes induced by phosphorylation therefore may illuminate a general mechanism by which receiver domains are activated.

V. Web Resources on NtrC and nitrogen metabolism

• http://plantbio.berkeley.edu/faculty/faculty_pages/Kustu.html
• http://nsm1.utdallas.edu/bio/Reitzer/Reitzer99.html
• http://miljolare.no/virtue/newsletter/01_03/curr-robb/index.php?utskrift=1
  

VI. References

1) Kern D, Volkman B., Luginbuhl P., Nohaile M., Kustu S., Wemmer D., (1999). Structure of a transiently phosphorylated switch in bacterial signal transduction. Nature, Vol 402, pp. 894-898.

2) Volkman et al, (1995). Three-Dimensional Solution Structure of the N-Terminal Receiver Domain of NTRC. Biochemistry, Vol 34, pp. 1413-1424.

3) Lee et al, (2001). Crystal structure of an activated response regulator bound to its target . Nature Structural Biology, January 2001, Vol 8, Number 1, pp. 53-56.

4) Branden & Tooze (1999). Intro to Protein Structure, Second Edition, pp. 56-57.

5) http://www.cchem.berkeley.edu/~wemmer/AbsProt/NTRC.html

6) Michael Nohaile, Dorothee Kern, David Wemmer, Kenneth Stedman, Sydney Kustu. Structural and Functional Analyses of Activating Amino Acid Substitutions in the Receiver Domain of NtrC: Evidence for an Activating Surface. Journal of Molecular Biology, Vol. 273, No. 1, Oct 1997, pp. 299-316

7) Cho, Seok-Yong Lee, Dalai Yan, Xiaoyu Pan, John S. Parkinson, Sydney Kustu, David E. Wemmer, Jeffrey G. Pelton (2000). Journal of Molecular Biology, Vol 297, No. 3, Mar 2000, pp. 543-551.

8) Halkides et al (2000). The 1.9A Resolution Crystal Structure of Phosphono-CheY, an Analogue of the Active Form of the Response Regulator, CheY, Biochemistry 2000, Vol 39, pp. 5280-5286.

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