Biophysical characterization of the domain association between cytosolic A and B domains of the mannitol transporter enzymes II^Mtl in the presence and absence of a connecting linker

Abstract

The mannitol transporter enzyme II^Mtl of the bacterial phosphotransferase system is a multi‐domain protein that catalyzes mannitol uptake and phosphorylation. Here we investigated the domain association between cytosolic A and B domains of enzyme II^Mtl, which are natively connected in Escherichia coli, but separated in Thermoanaerobacter tengcongensis. NMR backbone assignment and residual dipolar couplings indicated that backbone folds were well conserved between the homologous domains. The equilibrium binding of separately expressed domains, however, exhibited ∼28‐fold higher affinity compared to the natively linked ones. Phosphorylation of the active site loop significantly contributed to the binding by reducing conformational dynamics at the binding interface, and a few key mutations at the interface were critical to further stabilize the complex by hydrogen bonding and hydrophobic interactions. The affinity increase implicated that domain associations in cell could be maintained at an optimal level regardless of the linker.

Abbreviations

Ec

Escherichia coli

HSQC

heteronuclear single quantum correlation

IIB^Mtl

domain B of mannitol trasnporter enzyme II^Mtl

IIA^Mtl

domain A of mannitol trasnporter enzyme II^Mtl

ITC

isothermal titration calorimetry

K_D

equilibrium dissociation constant

PEP

phosphoenolpyruvate

PTS

phosphotransferase system

Tt

Thermoanaerobacter tengcongensis

Introduction

The phosphotransferase system (PTS) constitutes a central signaling network for sugar uptake and phosphorylation in bacteria.1, 2, 3 PTS is composed of two universal phosphotransferases, enzyme I, and HPr, and sugar‐specific transporters, enzymes II. Enzyme I transfers a phosphoryl group from phosphoenolpyruvate to the phosphocarrier protein HPr, and HPr delivers the phosphoryl group to enzymes II. Enzymes II are sugar‐specific transmembrane permeases comprising three or four domains that are linked or separated depending on bacterial strains and sugar specificity.4, 5 Enzyme II^Mtl is a mannitol‐specific transporter that is comprised of cytoplasmic IIA^Mtl and IIB^Mtl domains, and a transmembrane IIC^Mtl domain.6, 7 The phosphoryl group is sequentially transferred from HPr to IIA^Mtl, and then to IIB^Mtl, and finally to an incoming sugar molecule bound on IIC^Mtl. The structures of individual EcIIA^Mtl and EcIIB^Mtl domains of E. coli and their complex have been previously determined by x‐ray crystallography and NMR spectroscopy.8, 9, 10

Protein linkers are commonly observed in multi‐domain proteins to keep separate domains together and also to achieve domain motions for appropriate biological functions.11, 12 Flexible linkers, among others, effectively increase the local concentration of individual domains, enabling a stable complex formation between weakly interacting domains. Three domains of enzyme II^Mtl are expressed as a single polypeptide (IICBA^Mtl) in mesophilic gram‐negative bacteria, Escherichia coli, such that individual domains are connected by flexible linker residues.13 On the other hand, three domains are expressed as two separate polypeptides (IICB^Mtl and IIA^Mtl) in thermophilic gram‐negative bacteria, Thermoanaerobacter tengcongensis, such that B and C domains are covalently linked by a flexible linker, but A domain is expressed as a separate polypeptide (Supporting Information Fig. S1).14 It has been reported that EcIIA^Mtl and EcIIB^Mtl form a very weak complex with the equilibrium dissociation constant (K _D) of 3.7 mM.10 The intrinsically low affinity is compensated by a native connecting linker that substantially increases the effective local concentration to ∼4 mM.15 Calculation using the K _D value and the effective local concentration yield the associated state of the linked domains as ∼40%, which is comparable to the experimentally determined value of ∼30% from NMR spectroscopy.15

Since TtIIA^Mtl and TtIIB^Mtl of T. tengcongensis are separately expressed, the low affinity observed in their E. coli homologs would not allow them to associate, given the low cellular concentrations (10∼40 μM). In this study, we have examined the interaction between TtIIA^Mtl and TtIIB^Mtl using NMR spectroscopy and isothermal titration calorimetry (ITC). We show that individual domains of T. tengcongensis and E. coli adopt the same backbone folds, but separate domains exhibit a 28‐times higher affinity than the linked ones. The key binding determinants are identified, and the biological significances are discussed.

Results

Backbone structure and binding interfaces of the IIA^Mtl−IIB^Mtl complex of T. Tengcongensis

We cloned TtIIA^Mtl(1 − 146) and TtIIB^Mtl(386 − 477) based on the sequence alignment with the homologous EcIIA^Mtl(490 − 637) and EcIIB^Mtl(375 − 471). The active site His64 of TtIIA^Mtl and Cys395 of TtIIB^Mtl participated in the phosphoryl transfer reaction, but the phosphoryl groups on histidine and cysteine were labile and slowly lost to buffer by acid‐ or base‐catalyzed dephosphorylation reactions. When the active site Cys395 of TtIIB^Mtl was mutated to serine, TtIIB^Mtl(C395S) could be permanently phosphorylated as a stable form.16, 17 The 2D ¹H−¹⁵N HSQC spectra of TtIIA^Mtl, TtIIB^Mtl, and phospho‐TtIIB^Mtl(C395S) exhibited well‐isolated backbone amide resonances, indicating that they adopted a compact fold. Backbone chemical shifts of TtIIB^Mtl and phospho‐TtIIB^Mtl(C395S) were obtained using a suite of three‐dimensional heteronuclear correlation NMR experiments. Backbone chemical shifts of TtIIA^Mtl were obtained to completion based on previously results.18 We measured RDCs of backbone amide groups of TtIIA^Mtl, TtIIB^Mtl, and phospho‐TtIIB^Mtl(C395S) to examine whether they adopted the same backbone folds as their homologues in E. coli. RDCs were measured using 11 mg/ml of phage pf1 as an alignment media. For TtIIB^Mtl and phospho‐TtIIB^Mtl(C395S), 500 mM of NaCl was employed to prevent nonspecific interactions between TtIIB^Mtl and phage pf1. Comparison of observed and calculated ¹D_NH RDCs using the solution structures of EcIIA^Mtl and phospho‐EcIIB^Mtl(C384S) (PDB code 2FEW), and unphosphorylated EcIIB^Mtl (PDB code 1VKR) showed a good fitting with RDC R‐factors of 21.5%, 20.9%, and 20.7% for TtIIA^Mtl, TtIIB^Mtl, and phospho‐TtIIB^Mtl(C395S), respectively (Fig. 1). Our results indicate that backbone folds of individual domains of T. tengcongensis are largely the same as those of E. coli.

Figure 1.

RDC analysis of (A) TtIIA^Mtl, (B) TtIIB^Mtl, (C) phospho‐TtIIB^Mtl(C395S). Comparison of observed and calculated ¹D_NH RDCs obtained by SVD with the structures of EcIIA^Mtl (PDB code 2FEW) and EcIIB^Mtl (PDB code 1VKR) is shown. The RDCs were measured in an alignment medium of 11 mg/mL phage pf1. The values of RDC R‐factors and the correlation coefficients (r) are described. The magnitude of the principal component of the tensor ( $D_a^{\mathrm{N}\mathrm{H}}$), and the rhombicity (η) are as follows: −6.1 and 0.49 for TtIIA^Mtl, 7.9 and 0.25 for TtIIB^Mtl, and 5.8 and 0.28 for phospho‐TtIIB^Mtl(C395S).

We carried out NMR titrations to map the binding surfaces of the TtIIA^Mtl−TtIIB^Mtl complex. Chemical shift changes of backbone amide groups of ¹⁵N‐TtIIA^Mtl were monitored titrating with phospho‐TtIIB^Mtl(C395S). The chemical shift changes were mostly in the fast exchange regime on the chemical shift time scale, except for several residues (Arg49, Leu52, Thr55, Tyr69, Glu110, Ala112, Lys114, and Ile137) that exhibited line broadening due to an intermediate exchange. Residues with large chemical shift perturbation were observed in the α2−β2−β3, β5, α3, and α5 regions, which was consistent with the interaction surface of the EcIIA^Mtl [Fig. 2(A)]. We then monitored the backbone amide chemical shifts of ¹⁵N‐phospho‐TtIIB^Mtl(C395S) titrating with TtIIA^Mtl. Large chemical shift perturbations were observed in the β1−α1 loop, β2, and α2 regions, which corresponded to the binding interface of phospho‐EcIIB^Mtl(C384S) [Fig. 2(B)]. The chemical shift perturbation profiles indicated that interfaces of the TtIIA^Mtl−TtIIB^Mtl complex closely resembled those of the EcIIA^Mtl−EcIIB^Mtl complex.

Figure 2.

Chemical shift perturbation of (A) ¹⁵N‐TtIIA^Mtl titrated with phospho‐TtIIB^Mtl(C395S) and (B) ¹⁵N‐phospho‐TtIIB^Mtl(C395S) titrated with TtIIA^Mtl. Weighted ¹H_N/¹⁵N chemical shift perturbation (Δδ = ([(Δδ _HN)2 + (Δδ _N)²/25]/2)^1/2 is plotted as a function of resudue number. Secondary structures are shown above the sequence, and residues with line broadening during the titration was marked with X at the bottom. Multiple sequence alignment of (C) IIA^Mtl and (D) IIB^Mtl between Thermoanaerobacter tengcongensis, Escherichia coli, Yersinia pestis, Vibrio natriegens, and B. subtilis. Interfacial residues are marked with the asterisks, and the sequence similarity between T. tengcongensis and E. coli is color‐coded in red (identical), orange (similar) and cyan (not conserved).

Equilibrium domain−domain association between TtIIA^Mtl and TtIIB^Mtl

We measured the equilibrium binding constant between TtIIA^Mtl and TtIIB^Mtl using chemical shift perturbation and isothermal titration calorimetry. Nonlinear least squares optimization of the chemical shift perturbation data yielded the equilibrium dissociation constant (K _D) of 1.7 ± 0.2 mM between TtIIA^Mtl and TtIIB^Mtl, and 130 ± 20 μM between TtIIA^Mtl and phospho‐TtIIB^Mtl(C395S) [Fig. 3(A,B)]. The measured binding constant was fully consistent with the value of 130 ± 10 μM obtained by ITC [Fig. 3(C)]. The binding between TtIIA^Mtl and phospho‐TtIIB^Mtl(C395S) was a largely endothermic reaction with a favorable entropic contribution, which is typically observed for associations driven by hydrophobic interactions (Table 1). Previously, the K _D value between EcIIA^Mtl and EcIIB^Mtl was measured as 3.7 ± 0.2 mM, and the phosphorylation of EcIIB^Mtl did not significantly increase the binding affinity.10 Thus, the association between TtIIA^Mtl and unphosphorylated TtIIB^Mtl exhibited a modest two‐fold increase in the binding affinity compared to the EcIIA^Mtl−EcIIB^Mtl complex, whereas phosphorylation of TtIIB^Mtl enhanced the binding affinity by as much as ∼28 times.

Figure 3.

Backbone amide chemical shift perturbation upon titration of 0.3 mM ¹⁵N‐labeled TtIIA^Mtl with (A) TtIIB^Mtl and (B) phospho‐TtIIB^Mtl(C395S). The chemical shift changes were monitored at a spectrometer ¹H frequency of 600 MHz. All of the curves represent ¹H and ¹⁵N shift perturbations. The lines represent nonlinear least squares best fits to all of the titration data simultaneously using a simple equilibrium binding model. (C) Integrated heats of injections from the titration between TtIIA^Mtl and phospho‐TtIIB^Mtl(C395S). The squares are the experimental data and the lines represent the least‐squares best‐fit curve derived for one‐set of sites binding model. The thermodynamic parameters are given in Table I.

Table 1.

Equilibrium Dissociation Constants (K_D) Between IIA^Mtl and Phospho‐IIB^Mtl of E. coli and T. tengcongensis in Various Combinations Obtained by NMR Titration ^{a, b} and Isothermal Titration Calorimetry ^c

IIAMtl	IIBMtl	K D	ΔH	ΔS
		(μM)	(kcal/mol)	(cal/mol/deg)
EcIIAMtl	phospho‐EcIIBMtl(C384S)	3700 ± 200a
TtIIAMtl	TtIIBMtl	1700 ± 200a
TtIIAMtl	phospho‐TtIIBMtl(C395S)	130 ± 10c	6.7 ± 0.2	40.2
TtIIAMtl	phospho‐EcIIBMtl(C384S)	120 ± 30c	1.4 ± 0.1	22.5
EcIIAMtl	phospho‐TtIIBMtl(C395S)	N.D.a, b
Ec ${\text{IIA}}_{\mathrm{M}1}^{\text{Mtl}}$	phospho‐EcIIBMtl(C384S)	82 ± 20c	0.4 ± 0.1	19.9
Ec ${\text{IIA}}_{\mathrm{M}2}^{\text{Mtl}}$	phospho‐EcIIBMtl(C384S)	200 ± 60c	0.7 ± 0.1	19.4

^aDetermined by nonlinear least squares best fits to NMR titration data in 20 mM Tris, pH 7.4, at 25ºC (see Materials and Methods for detail).

^bNot determined due to the lack of chemical shift changes.

^cDetermined by ITC using one‐set of sites binding model in 20 mM Tris, pH 7.4, at 25ºC.

Identification of key interfacial residues responsible for the high affinity

To understand the driving forces that cause the large affinity increase, we compared the interfacial residues at the binding surface of the IIA^Mtl−IIB^Mtl complex between T. tengcongensis and E. coli. The binding interface has been well characterized between EcIIA^Mtl and phospho‐EcIIB^Mtl(C384S),10 and could be transferred to the TtIIA^Mtl−phospho‐TtIIB^Mtl(C395S) complex based on the sequence alignment [Fig. 2(C,D)]. EcIIA^Mtl and phospho‐EcIIB^Mtl(C384S) interacted using a wide and flat binding surface that formed key hydrophobic interactions assisted by weak electrostatic interactions. There were 27 residues at the interface of EcIIA^Mtl, and 18 residues at the interface of phospho‐EcIIB^Mtl(C384S).10 Between T. tengcongensis and E. coli, 8 residues were identical and 8 residues were similar at the interface of TtIIA^Mtl, and 16 residues were identical at the interface of TtIIB^Mtl [Fig. 2(C,D)]. The similarity scores at the interface were calculated as 59% and 89% for TtIIA^Mtl and TtIIB^Mtl, respectively, indicating that the interface of TtIIB^Mtl was particularly well conserved. The sequence similarity suggests that the affinity increase likely originated from the interface of TtIIA^Mtl. This assumption was supported by the measurement of cross‐strain binding affinity, where the K _D value between TtIIA^Mtl and phospho‐EcIIB^Mtl(C384S) was obtained as 120 ± 30 μM, which was comparable to the binding between TtIIA^Mtl and phospho‐TtIIB^Mtl(C395S) (Table 1). On the other hand, the cross‐strain binding between EcIIA^Mtl and phospho‐TtIIB^Mtl(C395S) was very weak and produced little chemical shift changes during the titration.

We further identified which interfacial residues of TtIIA^Mtl were responsible for the affinity increase. We note that TtIIA^Mtl associated with phospho‐TtIIB^Mtl(C395S) much stronger than with unphosphorylated TtIIB^Mtl, indicating that phosphorylation provided a key binding surface. It has been reported that phosphorylation entails a small shift in the active site backbone loop of EcIIB^Mtl, which stabilizes the phosphoryl group by a dense hydrogen bonding network.17 We focused on the interfacial residues of TtIIA^Mtl that provided intermolecular contacts with the phosphoryl group and also the surrounding loop region. Arg538, Thr542, and the active site His554 of EcIIA^Mtl interacted with the phosphoryl group of phospho‐EcIIB^Mtl(C384S). Arg538 made a salt bridge with the phosphoryl group, and His554 was directly involved in the phosphoryl transfer reaction. Thr542 interacted with the phosphoryl group and also with the loop region. Arg538 and His554 were highly conserved across most bacterial strains, whereas Thr542 was replaced by Ile52 in TtIIA^Mtl [Fig. 2(C)]. Residues that interacted with the active site loop surrounding the phosphoryl group were Thr542, Thr544, Gly555, Thr556, and V557 (Supporting Information Fig. S2). Among them, Thr544 and Gly555 were highly conserved across bacterial strains, whereas Thr542, Thr556, and Val557 were replaced by Ile52, Val66, and Ser67 in TtIIA^Mtl [Fig. 2(C)]. Thr542 interacted with Asp385 of EcIIB^Mtl, while Thr556 and Val557 interacted with Ala386 and Met388 of EcIIB^Mtl.

Based on the differences in the interfacial residues, we designed EcIIA^Mtl mutants with a higher affinity to phospho‐EcIIB^Mtl(C384S). The mutants were designed such that interfacial residues of EcIIA^Mtl were replaced by those of TtIIA^Mtl. The Ec ${\text{IIA}}_{\mathrm{M}1}^{\text{Mtl}}$ mutant contained two mutations at the end of the α2 helix (T542I/P543T), and the Ec ${\text{IIA}}_{\mathrm{M}2}^{\text{Mtl}}$ mutant contained two mutations (T556V/V557S) next to the active site His554. The equilibrium binding constants between EcIIA^Mtl mutants and phospho‐EcIIB^Mtl(C384S) were measured by ITC, and both mutants exhibited a significant increase in the binding affinity. Ec ${\text{IIA}}_{\mathrm{M}1}^{\text{Mtl}}$ showed 45 times stronger binding (K _D = 82 ± 23 μM), and Ec ${\text{IIA}}_{\mathrm{M}2}^{\text{Mtl}}$ showed 19 times stronger binding (K _D = 200 ± 60 μM) compared wild‐type EcIIA^Mtl. In addition, the backbone amide chemical shifts showed minor changes between wild‐type EcIIA^Mtl and the mutants, indicating that the backbone folds of the mutants were minimally perturbed (Supporting Information Fig. S3). To explain the structural basis for the increased affinity, we examined the interface of each EcIIA^Mtl mutant in complex with phospho‐EcIIB^Mtl(C384S). Based on the assumption that the backbone fold and binding interface of the EcIIA^Mtl mutants remained unaltered, one can speculate the favorable interactions arising from the mutants. Ec ${\text{IIA}}_{\mathrm{M}1}^{\text{Mtl}}$ achieved a high affinity mainly via hydrophobic cluster formation proximal to the active site loop. Ile542 of Ec ${\text{IIA}}_{\mathrm{M}1}^{\text{Mtl}}$ provided favorable hydrophobic interactions with Leu433 and Phe452 of phospho‐EcIIB^Mtl(C384S), which could reinforce the interaction of the α2 helix of Ec ${\text{IIA}}_{\mathrm{M}1}^{\text{Mtl}}$ with the active site loop of phospho‐EcIIB^Mtl(C384S) [Fig. 4(A)]. In Ec ${\text{IIA}}_{\mathrm{M}2}^{\text{Mtl}}$, the hydroxyl group of Ser557 of Ec ${\text{IIA}}_{\mathrm{M}2}^{\text{Mtl}}$ provided a hydrogen bond to the backbone carbonyl group of Ala386 of EcIIB^Mtl, which could strengthen the interaction of Arg538 and His554 of Ec ${\text{IIA}}_{\mathrm{M}2}^{\text{Mtl}}$ with the phosphoryl group of phospho‐EcIIB^Mtl(C384S) [Fig. 4(B)].

Figure 4.

The three dimensional structures of the binding interfaces between EcIIA^Mtl and phospho‐EcIIB^Mtl(C384S) (PDB code 2FEW). Left panels represent the interfaces of the EcIIA^Mtl−phospho‐EcIIB^Mtl(C384S) complex, and right panels represent the interfaces of (A) the Ec ${\text{IIA}}_{\mathrm{M}1}^{\text{Mtl}}$−phospho‐EcIIB^Mtl(C384S) complex, and (B) the Ec ${\text{IIA}}_{\mathrm{M}2}^{\text{Mtl}}$−phospho‐EcIIB^Mtl(C384S) complex. Interfacial residues are annotated amd mutations sites of the EcIIA^Mtl mutants are labeld in red. Interfacial residues of phospho‐EcIIB^Mtl(C384S) are labeled in italic type. The hydrophobic cluster formed by I542 of Ec ${\text{IIA}}_{\mathrm{M}1}^{\text{Mtl}}$, and L433 and F452 of phospho‐EcIIB^Mtl(C384S) is highlighted by a yellow shade. The hydrogen bonding formed by S557 of Ec ${\text{IIA}}_{\mathrm{M}2}^{\text{Mtl}}$ and A386 of phospho‐EcIIB^Mtl(C384S) is highlighted by a thick dashed line.

Discussion

Many proteins exist as multi‐domain proteins that are connected by linkers of variable lengths and sequences. When there is a gain or loss of the linker due to environmental pressure, the equilibrium domain−domain association would be considerably altered. In this study, we showed that minimal mutations at the domain interface were sufficient to maintain the domain associations of Enzyme II^Mtl in response to gain or loss of the linker. Cellular concentrations of TtIIA^Mtl and TtIIB^Mtl have not been experimentally determined, but can be roughly estimated as ∼40 μM and ∼10 μM, respectively, from the reported values of the glucose transporter Enzyme II^Glc.19 The estimated concentrations and the measured K _D value (130 μM) predict that phosphorylated TtIIB^Mtl would associate with TtIIA^Mtl ∼23% of the time, which is remarkably close to ∼30% of the associated state observed for EcIIA^Mtl and EcIIB^Mtl. Thus, the affinity increase effectively compensates for the loss of the linker, enabling domain associations in cell at an optimal level regardless of the linker. It is not clear what caused TtIIA^Mtl to disconnect from TtIIB^Mtl while EcIIA^Mtl and EcIIB^Mtl were covalently linked. We postulate that TtIIA^Mtl may participate in another metabolic signaling pathway that is regulated by mannitol uptake and phosphorylation.

We note that the affinity increase between TtIIA^Mtl and TtIIB^Mtl was subject to protein phosphorylation. Phosphorylation of PTS proteins regulates diverse cellular signaling pathways of bacterial carbon metabolism.20 It has been reported that phosphorylation induces large changes in the conformational states, stability, and dynamics of Enzyme I, which regulates the initiation of phosphoryl transfer reactions.21, 22, 23, 24, 25 On the other hand, impact of phosphorylation on equilibrium association appeared to be less pronounced between Enzyme I, HPr, and Enzymes II of E. coli.10, 26, 27, 28, 29 We show that the interaction between TtIIA^Mtl and TtIIB^Mtl was highly sensitive to the phosphorylation state. Increased binding affinity upon phosphorylation would be beneficial for rapid association for phosphoryl transfer reactions, suppressing futile associations between unphosphorylated proteins. It has been reported that protein phosphorylation causes relatively large changes in binding energy for one‐third of protein complexes in human proteome.30 The correlation between phosphorylation and binding energy, however, is not currently well understood. Our study suggests that the presence of a linker may be one of the factors that influence the correlation between phosphorylation and equilibrium binding energy.

We have identified the key interfacial residues of TtIIA^Mtl that contributed to the increased binding affinity of the TtIIA^Mtl−phospho‐TtIIB^Mtl(C395S) complex compared to the EcIIA^Mtl−phospho‐EcIIB^Mtl(C384S) complex. We showed that phosphorylation was important to the increased affinity, but the identified interfacial residues did not participate in direct intermolecular contact with the phosphoryl group. Instead, phosphorylation of TtIIB^Mtl altered the conformational dynamics of the active site β1−α1 loop region that constituted the main binding interface for TtIIA^Mtl. In the 2D ¹H−¹⁵N HSQC spectrum of unphosphorylated TtIIB^Mtl, residues in the active site loop region were largely broadened out due to a conformational exchange. Specifically, the backbone amide groups of Asp396, Ala397, Gly398, Met399, Gly400, and Ser402 that formed the active site P loop to stabilize the phosphoryl group were not observed in unphosphorylated TtIIB^Mtl. The conformational exchange mostly disappeared upon phosphorylation, as the loop residues engaged in multiple hydrogen bonds to the phosphoryl group (Supporting Information Fig. S4). Thus, phosphorylation of TtIIB^Mtl seems to effectively suppress the conformational dynamics at the active site loop, providing a well‐ordered binding interface for TtIIA^Mtl.

Materials and Methods

Sample preparation

The coding regions were PCR‐amplified and cloned into a modified pET32a vector.10 Mutation of the active site and the interfacial residues were introduced by QuikChange (Agilent Technologies, Inc.), and the new constructs were verified by DNA sequencing. The expression vectors were transformed into E. coli strain BL21star(DE3) (Invitrogen), and the cells were grown in Luria Bertani or minimal media (with ¹⁵NH₄Cl and/or ¹³C₆‐glucose as the sole nitrogen or carbon source, respectively). Proteins were overexpressed and purified as previously described. Phosphorylation of TtIIB^Mtl or EcIIB^Mtl was conducted as follows: 100 μM IIB^Mtl was incubated overnight with 5 mM MgCl₂, 5 mM phosphoenolpyruvate, 1 μM Enzyme I, 2 μM HPr, and 1 μM IIA^Mtl in 20 mM Tris, pH 7.4. EcIIB^Mtl(C384S) was phosphorylated at 37°C, and TtIIB^Mtl(C395S) was phosphorylated at 65°C. The phosphorylation reaction was monitored by native gel electrophoresis, and purified by monoQ anion exchange chromatography (GE Healthcare). Phosphorylation was confirmed by mass spectrometry and 2D heteronuclear single quantum correlation (HSQC) NMR spectra. Purified proteins were finally concentrated in 20 mM Tris, pH 7.4.

NMR spectroscopy

NMR spectra were collected at 25ºC on Bruker AVANCE 600 MHz and 700 MHz spectrometers equipped with either a z‐shielded gradient triple resonance cryoprobe or an x,y,z‐shielded probe. Sequential assignments of TtIIB^Mtl, phospho‐TtIIB^Mtl(C395S), and TtIIA^Mtl were performed using 3D triple resonance experiments such as HNCO, HN(CA)CO, CBCA(CO)NH, and HNCACB. NMR Spectra were processed using the NMRPipe program31 and analyzed using the PIPP³² and NMRView programs.33 For NMR titration measurement, 0.3 mM ¹⁵N‐labeled samples were titrated with unlabeled partner proteins up to a 1:24 molar ratio. Measured ¹H and ¹⁵N chemical shift changes were weighted using the equation Δδ _HN = [(Δδ _H ² + 0.04 Δδ _N ²)/2]^1/2. The titration data were fit to a simple equilibrium binding model to yield the equilibrium dissociation constant (K _D) using the equation, Δδ _obs = Δδ _max* $\frac{\left\{\left(\left[A\right]+\left[B\right]+K_D\right)- {[{\left(\left[A\right]+\left[B\right]+K_D\right)}^2-\left(4\left[A\right]\left[B\right]\right)]}^{1/2}\right\}}{2\left[A\right]}$, where Δδ _obs is the observed chemical shift change, Δδ _max is the maximum chemical shift change, and [A] and [B] are the total protein concentrations of the titrate and the titrant, respectively. ¹D_NH residual dipolar couplings (RDCs) were measured by taking the difference in ¹J_NH scalar couplings in aligned and isotropic media.34, 35 The alignment media employed were phage pf1 (ASLA Biotech),36, 37 and singular value decomposition (SVD) analysis of RDCs was carried out using Xplor‐NIH.38

Isothermal Titration Calorimetry

ITC was performed using an iTC₂₀₀ calorimeter (Malvern Instrument). 0.5 mM TtIIA^Mtl was placed in the cell and titrated with 5 mM phospho‐TtIIB^Mtl(C395S) in the syringe at 25°C for wild‐type and mutant proteins. Titrations was employed as twenty consecutive 2 μL aliquots of phospho‐ TtIIB^Mtl(C395S) into the cell containing TtIIA^Mtl with intervals of 180 sec. Analysis of the data was performed using the Origin software provided with the instrument.

Supporting information

Supporting Information Figure 1

Supporting Information Figure 2

Supporting Information Figure 3

Supporting Information Figure 4