Alanine Scanning Mutagenesis of Anti-TRAP (AT) Reveals Residues Involved in Binding to TRAP

Chen, Yanling; Gollnick, Paul

doi:10.1016/j.jmb.2008.02.015

Cited by 8 publications

(22 citation statements)

References 50 publications

(9 reference statements)

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“…Chen and Gollnick reconcile their model with these data by suggesting that the 4 AT trimers do indeed bind in this way. Their model, however, shows the AT trimers to be widely spaced, with no direct contact, and offers no explanation of the considerable cooperativity reportedly required by the AUC data (20). Our model places the Glu-22A side-chain of 1 AT trimer within salt-bridging distance of the N-terminal nitrogen atom of Met-1C of the adjacent AT trimer.…”

Section: Discussionmentioning

confidence: 68%

“…However, the model they present is based merely on bringing Val-2, Ile-3, Asp-6, and Asp-7 into proximity with Lys-37, Lys-56, and Arg-58, and offers no atomic detail. In the simple space-filling model presented (20), it appears the rotation axis of AT lies perpendicular to that of TRAP, and 1 11-mer TRAP ring may bind up to 4 AT trimers because of steric hindrance. The mass spectrometry results shown in Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Val-2, Ile-3, and Asp-7 play an indirect role in maintaining the structure of the N terminus of AT so that Met-1 can interact strongly with TRAP. Putting either an alanine or leucine codon at position 2 of the AT gene abolishes binding of the polypeptide product to TRAP (20). Replacing Val-2 with alanine, however, will allow Escherichia coli methionine aminopeptidase to remove Met-1, which the crystal structures show to be key to binding, although Chen and Gollnick (20) did not apparently examine this possibility.…”

Section: Discussionmentioning

confidence: 99%

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The nature of the TRAP–Anti-TRAP complex

Watanabe

Heddle

Kikuchi

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Tryptophan biosynthesis is subject to exquisite control in species of Bacillus and has become one of the best-studied model systems in gene regulation. The protein TRAP (trp RNA-binding attenuation protein) predominantly forms a ring-shaped 11-mer, which binds cognate RNA in the presence of tryptophan to suppress expression of the trp operon. TRAP is itself regulated by the protein Anti-TRAP, which binds to TRAP and prevents RNA binding. To date, the nature of this interaction has proved elusive. Here, we describe mass spectrometry and analytical centrifugation studies of the complex, and 2 crystal structures of the TRAP-Anti-TRAP complex. These crystal structures, both refined to 3.2-Å resolution, show that Anti-TRAP binds to TRAP as a trimer, sterically blocking RNA binding. Mass spectrometry shows that 11-mer TRAP may bind up to 5 AT trimers, and an artificial 12-mer TRAP may bind 6. Both forms of TRAP make the same interactions with Anti-TRAP. Crystallization of wild-type TRAP with Anti-TRAP selectively pulls the 12-mer TRAP form out of solution, so the crystal structure of wild-type TRAP-Anti-TRAP complex reflects a minor species from a mixed population.protein complex ͉ X-ray crystallography ͉ transcription regulation ͉ analytical ultracentrifugation ͉ mass spectrometry T RAP (trp RNA-binding attenuation protein) plays a central role in the highly intricate regulation of transcription and translation of the trp operon in several species of Bacillus, and this system has provided important insights into several mechanisms of gene regulation (1-5). The protein forms an 11-mer ring that binds 11 molecules of tryptophan (Trp) at symmetryrelated sites (6-8), and the TRAP-Trp-RNA complex has been solved for the Bacillus stearothermophilus protein (9). With tryptophan bound, TRAP can bind trp mRNA and induce transcription termination. In Bacillus subtilis, when levels of charged tRNA Trp fall, the protein Anti-TRAP (AT) is expressed, which blocks RNA binding to Trp-bound TRAP and relieves expression of the trp operon (10, 11). AT forms a stable trimer, 4 copies of which can further associate into a dodecamer form (12, 13). Each 53-residue polypeptide chain carries a zincbinding site made of 4 cysteine residues, and chemical crosslinking studies suggested that the active form of AT may be a hexamer (14). Analytical ultracentrifugation data have been interpreted to indicate that the AT dodecamer is the active (TRAP-binding) form, giving an AT 12 -TRAP 11 complex, although it was noted that the data did not rule out alternative stoichiometry (12). Antson and colleagues (13) further proposed, on the basis of their X-ray structure of AT, a model in which the AT dodecamer may bind up to 4 TRAP 11-mer rings by aligning their 11-fold symmetry axes with its 3-fold axes. In this model, AT would not directly block RNA binding, or contact TRAP residues Lys-37 and Arg-58 that are known to be required for TRAP-RNA (15) and TRAP-AT interaction (10). Instead it penetrates the TRAP ring, and it is suggested that there is indirect ...

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Section: Discussionmentioning

confidence: 68%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

The nature of the TRAP–Anti-TRAP complex

Watanabe

Heddle

Kikuchi

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…Because structural and biochemical data indicate that only trimeric AT can bind and inhibit TRAP (21,26,27), a cellular shift to low pH would favor AT 12 formation and result in decreased gene expression. The coincidence of the internal pH of growing B. subtilis cells with our measured pK a for deprotonation of the amino terminus of AT 3 (pH 7.4) indicates that changes in cellular pH could alter the trimer-dodecamer equilibrium and thereby alter gene expression profiles.…”

Section: Discussionmentioning

confidence: 99%

“…Site-directed mutations in TRAP that interfere with RNA binding were found to also interfere with AT binding, suggesting that AT may bind to the RNA-binding surface of TRAP that is stabilized by tryptophan binding (22)(23)(24)(25). Alanine scanning studies of AT highlighted residues important for TRAP binding and showed that dodecameric AT was unlikely to be able to bind to TRAP (26). In agreement with these findings, crystallographic studies of 12-mer-forming variants of TRAP in complex with AT showed six AT trimers (AT 3 ) arranged along the outside of the TRAP ring, suggesting that the trimer is the species that binds to TRAP (27).…”

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confidence: 99%

Mechanism for pH-dependent gene regulation by amino-terminus-mediated homooligomerization of Bacillus subtilis anti- trp RNA-binding attenuation protein

Sachleben

McElroy

Gollnick

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Anti-TRAP (AT) is a small zinc-binding protein that regulates tryptophan biosynthesis in Bacillus subtilis by binding to tryptophanbound trp RNA-binding attenuation protein (TRAP), thereby preventing it from binding RNA, and allowing transcription and translation of the trpEDCFBA operon. Crystallographic and sedimentation studies have shown that AT can homooligomerize to form a dodecamer, AT 12 , composed of a tetramer of trimers, AT 3 . Structural and biochemical studies suggest that only trimeric AT is active for binding to TRAP. Our chromatographic and spectroscopic data revealed that a large fraction of recombinantly overexpressed AT retains the N-formyl group (fAT), presumably due to incomplete N-formyl-methionine processing by peptide deformylase. Hydrodynamic parameters from NMR relaxation and diffusion measurements showed that fAT is exclusively trimeric (AT 3 ), while (deformylated) AT exhibits slow exchange between both trimeric and dodecameric forms. We examined this equilibrium using NMR spectroscopy and found that oligomerization of active AT 3 to form inactive AT 12 is linked to protonation of the amino terminus. Global analysis of the pH dependence of the trimer-dodecamer equilibrium revealed a near physiological pK a for the N-terminal amine of AT and yielded a pH-dependent oligomerization equilibrium constant. Estimates of excluded volume effects due to molecular crowding suggest the oligomerization equilibrium may be physiologically important. Because deprotonation favors "active" trimeric AT and protonation favors "inactive" dodecameric AT, our findings illuminate a possible mechanism for sensing and responding to changes in cellular pH.allostery | thermodynamic linkage | n-formyl methionine I n bacilli, the trp RNA-binding attenuation protein (TRAP) is responsible for responding to intracellular levels of tryptophan and regulating transcription (1-4) and translation (5-10) of six tryptophan biosynthetic genes clustered in the trpECDFBA operon (1, 11). Transcription of the gene is controlled by an attenuation mechanism, involving competing terminator and antiterminator RNA structural elements in the 5′ leader region of the mRNA. In the absence of cellular tryptophan, the antiterminator structure is favored, allowing transcription and translation of the operon. When cellular tryptophan levels are high, the amino acid binds to TRAP, which becomes activated for binding to an RNA site that overlaps the antiterminator sequence, favoring formation of the terminator; translation of these genes is regulated by a similar mechanism (1,6,9,10,(12)(13)(14). TRAP is a small protein (∼74 amino acids) that assembles into an undecameric (11-mer) ring-shaped structure, with binding sites for tryptophan arranged between neighboring TRAP protomers (15, 16). RNA binds to TRAP by wrapping around the outside of this ring (17).In Bacillus subtilis (Bsu), the protein anti-TRAP (AT), encoded by the yczA gene, provides further regulation by inhibiting mRNA binding by tryptophan-activated TRAP (13,18). AT is a zinc-bind...

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Bacillus licheniformis Anti-TRAP can assemble into two types of dodecameric particles with the same symmetry but inverted orientation of trimers

Shevtsov

Chen

Isupov

et al. 2010

Journal of Structural Biology

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Anti-TRAP (AT) protein regulates expression of tryptophan biosynthetic genes by binding to the trp RNA-binding attenuation protein (TRAP) and preventing its interaction with RNA. Bacillus subtilis AT forms trimers that can either interact with TRAP or can further assemble into dodecameric particles. To determine which oligomeric forms are preserved in AT proteins of other Bacilli we studied Bacillus licheniformis AT which shares 66% sequence identity with the B. subtilis protein. We show that in solution B. licheniformis AT forms stable trimers. In crystals, depending on pH, such trimers assemble into two different types of dodecameric particles, both having 23 point group symmetry. The dodecamer formed at pH 6.0 has the same conformation as previously observed for B. subtilis AT. This dodecamer contains a large internal chamber with the volume of ∼700 Å3, which is lined by the side chains of twelve valine residues. The presence of the hydrophobic chamber hints at the possibility that the dodecamer formation could be induced by binding of a ligand. Interestingly, in the dodecamer formed at pH 8.0 all trimers are turned inside out relatively to the form observed at pH 6.0.

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Alanine Scanning Mutagenesis of Anti-TRAP (AT) Reveals Residues Involved in Binding to TRAP

Cited by 8 publications

References 50 publications

The nature of the TRAP–Anti-TRAP complex

The nature of the TRAP–Anti-TRAP complex

Mechanism for pH-dependent gene regulation by amino-terminus-mediated homooligomerization of Bacillus subtilis anti- trp RNA-binding attenuation protein

Bacillus licheniformis Anti-TRAP can assemble into two types of dodecameric particles with the same symmetry but inverted orientation of trimers

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