How Integration of Positive and Negative Regulatory Signals by a STAND Signaling Protein Depends on ATP Hydrolysis  Emélie Marquenet, Evelyne Richet  Molecular Cell  Volume 28, Issue 2, Pages 187-199 (October 2007) DOI: 10.1016/j.molcel.2007.08.014 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 The D129A Substitution Abolishes the ATPase Activity of MalT (A) Domain structure. NBD, HD, and WHD are the nucleotide binding domain, the helical domain, and the winged helix domain, respectively. The domain following WHD is as of yet ill defined. The sensing domain comprises eight SUPR-type amino acid repeats. The effector domain is a LuxR-type DNA-binding domain. (B) Sequence alignment of the extended Walker B regions of STAND MalT subfamily proteins. (C) Effect of the D129A substitution on MalT ATPase activity. ATPase assays were performed in the presence of 2 μM His-tagged protein, in the absence and in the presence of 1 mM maltotriose. Molecular Cell 2007 28, 187-199DOI: (10.1016/j.molcel.2007.08.014) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 In Vivo Activity of MalT Variants Altered in the Walker B Region (A) Levels of β-galactosidase in malT derivatives of isogenic ΔmalK (no repressors), malK+ (MalK+ MalY−), and ΔmalK malI::Tn10 (MalK− MalY+) reporter strains (pop7175, pop7255, and pop7186, respectively) grown in minimal medium + glycerol. The values are the means ± SD from at least two independent experiments. (B) As in (A), except that the bacteria were grown in the presence of glucose, instead of glycerol. The strong decrease in the level of β-galactosidase observed is due to the fact that the glucose effect reduces not only malT expression but also the activity of malEpΔ92, the promoter assayed, through the CRP site present in malEpΔ92 (Vidal-Ingigliardi et al., 1991). (C) Immunoblots. Total cell extracts from pop7175 and malT derivatives thereof grown in the presence of glycerol were analyzed by SDS-PAGE and developed with anti-MalT and anti-σ70 antibodies. The relative levels of MalT were normalized with respect to the levels of σ70. The values are the means ± SD of the relative levels obtained with three lanes loaded with the same extract. Molecular Cell 2007 28, 187-199DOI: (10.1016/j.molcel.2007.08.014) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 In Vitro Properties of MalT-D129A (A) MalT self-association in the presence of ATP and maltotriose. (His)6-MalT (20 μM) was preincubated and filtered through a Superdex 200 column in the presence of 0.1 mM ATP and 1 mM maltotriose. The retention volumes (ml) of the peaks are indicated. (B) MalT self-association in the presence of maltotriose alone. As in (A), except that ATP was omitted throughout the experiment. (C) MalT self-association in the absence of effectors. As in (A), except that both effectors were omitted throughout the experiment. (D) Activation of open-complex formation. Abortive initiation assays were performed with His-tagged proteins in the presence of 50 μM ATP and 1 mM maltotriose. The sigmoidal curves reflect cooperative binding of MalT to the four MalT sites present in the promoter used as a template. (E) MalT affinity for maltotriose. Abortive initiation assays were performed in the presence of 50 μM ATP, the indicated concentration of maltotriose, and 30 nM wild-type (His)6-MalT or 106 nM (His)6-MalT-D129A. Molecular Cell 2007 28, 187-199DOI: (10.1016/j.molcel.2007.08.014) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Effect of the D129A Substitution on MalT Interaction with MalY (A) MalY (5 μM) alone, wild-type (His)6-MalT (10 μM) alone, or wild-type (His)6-MalT (10 μM) + MalY (5 μM) was preincubated and filtered through a Superdex 200 column in the presence of 0.1 mM ATP and 10 μM PLP. The absorbance at 390 nm, which monitors PLP, the MalY cofactor, is multiplied by a factor of 9. (B) As in (A), with (His)6-MalT-D129A instead of wild-type (His)6-MalT. The dashed lines represent the protein peaks obtained with MalT-D129A and MalY, when filtered separately. Molecular Cell 2007 28, 187-199DOI: (10.1016/j.molcel.2007.08.014) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 The Exchange of ADP for ATP Depends on Maltotriose (A) Nontagged MalT (1.8 μM; 180 pmoles) was incubated alone or in the presence of 500 μM AMP-PNP ± 20 μM maltotriose and passed through a centrifuge column to remove unbound ligands and assay the amount of ADP bound to MalT. The values are the means ± SD from two independent experiments performed in duplicates. (B) (His)6-MalT (2 μM, 200 pmoles) was incubated in the presence of 20 μM [α-32P]ATP ± 1 mM maltotriose and passed through a centrifuge column to remove unbound nucleotides and determine the amount of radioactive nucleotide bound to the protein. The values are the means ± SD from at least two independent experiments. (C) As in (B), except that [α-32P]ATP was replaced with [γ-32P]ATP. (D) Apparent initial rates of ATPγS binding by wild-type MalT and MalTc26. Vi was measured in the presence of 2 μM untagged MalT, 1 μM [35S]ATPγS, and maltotriose as indicated. Molecular Cell 2007 28, 187-199DOI: (10.1016/j.molcel.2007.08.014) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 The MalT Cycle The circles and pentagons represent ATP-bound and ADP-bound forms of MalT in open conformation, i.e., able to multimerize upon maltotriose binding, albeit with a much reduced efficiency for the latter. The square represents resting MalT, a MalT/ADP form in “closed” conformation, that would be similar to the crystallized form of ADP-bound ΔWD40 Apaf-1 (Riedl et al., 2005). In vivo, ATP hydrolysis may occur only at the level of the nucleoprotein complexes if MalT self-associates solely upon promoter binding. Molecular Cell 2007 28, 187-199DOI: (10.1016/j.molcel.2007.08.014) Copyright © 2007 Elsevier Inc. Terms and Conditions