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Dimerization and Conformational Exchanges of the Receiver Domain of Response Regulator PhoB From Escherichia Coli Xinhui Kou, Yixiang Liu, Conggang Li, Maili Liu, and Ling Jiang J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b01034 • Publication Date (Web): 03 May 2018 Downloaded from http://pubs.acs.org on May 5, 2018

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The Journal of Physical Chemistry

Dimerization and Conformational Exchanges of the Receiver Domain of Response Regulator PhoB from Escherichia coli Xinhui Kou1, 2 ·Yixiang Liu1·Conggang Li1·Maili Liu1·Ling Jiang*1 1

Key Laboratory of Magnetic Resonance in Biological Systems, National Center for Magnetic

Resonance in Wuhan, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, the Chinese Academy of Sciences, Wuhan 430071, China 2

Graduate University of Chinese Academy of Science, 19A Yuquanlu, Beijing 100049, China

*Corresponding author: [email protected].

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Abstract PhoB is a response regulator of PhoR/PhoB two-component system from Escherichia coli which is involved in the environmental phosphate regulation. It has been reported that the N-terminal receiver domain (PhoBN) forms a dimer using the α1-α5 face in the apo state and a dimer using the α4-β5-α5 face in the active state investigated by X-ray crystallography. However, it is not clear whether the conformational switch of the dimer is dependent on phosphorylation. Here, we report the NMR studies of PhoBN in solution in its apo form. We observed that the secondary structural fragments of apo PhoBN characterized by NMR are almost the same as those determined by crystallography, but the NMR spectrum of PhoBN shows inhomogeneous amide signals. Concentration dilution experiments and backbone relaxation parameters showed that PhoBN exists in equilibrium between monomer and dimer states. Using paramagnetic relaxation enhancement experiments, we demonstrated that the dimer of apo PhoBN forms several transient dimer interfaces in solution. This finding suggested that, in addition to the monomer-to-dimer exchange, the inactive conformation of PhoBN has different domain arrangements which are independent of phosphorylation. It provides an experimental data for the conformational selection mechanism of the phosphorylation of PhoBN.


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Introduction Two-component systems (TCSs) are cellular signaling systems which exit in prokaryotic bacterial cells and a limited number of organisms (fungi, slime molds and plants)

1,2

, but not in animals and

humans. TCSs control many bacterial adaptive behaviors and pathways such as environmental stress response, central metabolism, chemotaxis, virulence and pathogenesis, making themselves attractive drug targets. The simplest TCS

3,4

consists of two proteins, a histidine kinase (HK)

5

and a response

regulator (RR) 6. Most of the response regulators have two functional domains, the N-terminal receiver domain and the C-terminal effector domain. The receiver domain of most RR proteins shows a conserved β5α5 sandwich-structured conformation and contains a highly conserved aspartate residue that can be phosphorylated by its cognate histidine kinase. The phosphorylation induces conformational change of the full-length RR that triggers the interaction between effector domain and the downstream gene or protein targets for cellular regulation. The majority of response regulators can be classified into groups termed as OmpR/PhoB, FixJ/NarL, and NtrC/DctD subfamilies, based on the similarities in their effector domains. The OmpR/PhoB family is the largest one that covers about 45% of the RRs 7. To date, crystal structures of seven full-length proteins in OmpR/PhoB family have been reported, such as DrrB and DrrD from Thermotoga maritime

8,9

, RegX3, MtrA, PrrA and PhoP from mycobacterium tuberculosis

10-13

and

BaeR from Escherichia coli 14. Among these proteins, dimerization or higher-order oligomerization is commonly observed and highly correlated with their functions. In most cases, the dimerization is mediated by their receiver domains. For instance, dimerization was observed for the phosphorylated full-length DrrB, while the isolated receiver domain of DrrB shows a monomer to dimer transition between its unphosphorylated and phosphorylated states, suggesting that the receiver domain alone is



Page 4 of 31

sufficient for the dimerization 9. MtrA is a monomer in unphosphorylated state and the α4-β5-α5 face of the receiver domain is stabilized by intra-molecular interactions with its effector domain. In the active state of MtrA, the α4-β5-α5 face is disrupted and forms inter-molecular interactions that is proposed to mediate dimerization 11. The apo PhoP from Mycobacterium tuberculosis, forms a dimer in crystal structure through the α4-β5-α5 face of the receiver domain, and the phosphorylation facilitates the dimerization further to increase the DNA binding affinity of the effector domain

13

. The crystal

structure of the unphosphorylated BaeR from Escherichia coli forms a dimer as well, but the dimer interface is formed by a domain swap between the β-core domain and α4-β5 elements of the receiver domain, and helix α4 is completely unwound in both promoters

14

. Domain swapping has also been

reported in the active structure of RegX3, which is stabilized by a unique three-dimensional domain swapping of α4-β5-α5 elements of receiver domain

12

. Inactive state of RegX3 adopts a reasonably

compact conformation, and exists as both a monomer and a dimer in a concentration-dependent manner 15

. Therefore, most of the response regulators in the OmpR/PhoB family form dimers mediated by

α4-β5-α5 face of the receiver domain, in a conformational equilibrium between inactive and active states, and the equilibrium is shifted toward the active state upon phosphorylation. Some response regulators which cannot be phosphorylated can also form homodimer through the α4-β5-α5 dimer interface in apo form in the OmpR/PhoB family such as GlnR from Amycolatopsis mediterranei 16. PhoB is a response regulator protein from Escherichia coli which belongs to the largest OmpR/PhoB family. It mediates the responses to changing concentrations of the environmental phosphate Pi signal, which is essential for bacterial growth

17,18

. The crystal structures of active and

inactive receiver domain of PhoB (PhoBN) have been reported, respectively 19,20. It shows a conserved β5α5 folding, forming a homodimer with an α1-α5 face in the apo form and an α4-β5-α5 dimer in the


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phosporylated form

19

. The structural information reveals that dimerization is the common feature of

PhoBN both in the inactive and active states, however, the α1-α5 dimerization interface may not be physiologically important

21

and it is rarely observed in other RRs. Additionally, whether the dimer

interface switch is dependent on phosphorylation remains unclear. Here we analyzed the structural features of apo PhoBN in solution by using nuclear magnetic resonance spectroscopy (NMR). The backbone resonances of PhoBN have been assigned by using three-dimensional NMR experiments with 1

H,

15

N,

13

C-labeled protein. Dilution experiments (from 1.42 mM to 0.17 mM) of PhoBN by 1H-15N

HSQC spectra reveal a monomer to dimer exchange. Paramagnetic relaxation enhancement (PRE) measurements show that apo PhoBN has dimer arrangement exchanges mediated by multiple interfaces and these results provide basic information for better understanding of the phosphorylation process of response regulators.

Experimental methods Sample Preparation The E.coli phoB gene was cloned into the pET-28a vector (Novagen) using the NdeI and XhoI restriction enzyme sites to express PhoBN (1~125 residue) protein with thrombin-cleavable His6 tag 22 at the N-terminus. The constructed plasmid was transformed into E.coli BL21(DE3) stain for protein expression and purification. The expression cell stock was grown in LB medium containing 50 ug/mL kanamycin at 37℃ overnight. 15N-labeled and 13C-labeled PhoBN was expressed in 500 mL M9 media supplemented with 0.5 g

15

NH4Cl and 1.0 g

13

C-glucose (Cambridge Isotope Laboratories, Andover, MA, USA). When

OD600 of the culture reached to 0.7~0.8, the cells were induced by 1.0 mM isopropyl thio



β-D-1-galactopyranoside at 20 ℃ for 20-21 hours before harvested. The cultured cells were resuspended in lysate buffer (50 mM Tris, 300 mM NaCl, and 20 mM imidazole, pH 7.5), and disrupted by sonication on ice. The cell debris was removed by centrifugation (48384 ×g, 40 min, 4 ℃). Liquid supernatant was filtrated and then loaded onto the Ni2+-NTA column which was equilibrated with buffer A (50 mM Tris, 300 mM NaCl, and 20 mM imidazole, pH 7.5). PhoBN was eluted by 70 % (v/v) buffer B (50 mM Tris, 300 mM NaCl, and 500 mM imidazole, pH 7.5). The His6-fused protein was cleaved by bovine thrombin (2-3 hours, room temperature). After thrombin cleavage, the resultant protein contained an additional Gly-Ser-His at the N terminus. The redundant bovine thrombin was removed by benzamidine column. After that, the PhoBN protein was loaded onto Ni2+-NTA column again to remove the uncleaved protein and then further purified by Superdex 75 (buffer containing 50 mM Tris, 100 mM NaCl, and 5 mM DTT, pH 8.0) and desalting column. The plasmid of mutation PhoBNC19A and other mutations were produced by PCR and the expression and purification protocols were the same as those of wt-PhoBN . NMR experiments All the NMR samples were prepared in the buffer containing 20 mM HEPES (pH 7.0), 100 mM NaCl, 5 mM DTT, 10% (v/v) D2O and 90% (v/v) H2O. NMR experiments were performed on 700MHz bruker spectrometer equipped with cryogenic triple-resonance probes with 1H/13C/15N z-pulsed magnetic field gradient at 298K, and all the data were processed by Topspin3.2 and NMRpipe 23,24. NMR backbone resonance assignment of apo PhoBN: A standard set of multiple resonance NMR experiments including 2D 1H-15N HSQC, 2D 1H-13C HSQC, 3D HNCACB, 3D CBCA(CO)NH, 3D HNCO, 3D HN(CO)CA, and 3D HNCA 25 were recorded. Protein concentration was about 0.5 mM. Backbone assignments were analyzed with CARA (http://cara.nmr.ch/doku.php). Secondary structure


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elements were predicted from assigned chemical shifts (13Cα and 13Cβ ) using the TALOS+ program 26. 15

N backbone relaxation measurements: The longitudinal (T1) spin-relaxation rates of PhoBN

were measured with relaxation delays of 0.002, 0.120, 0.250, 0.450, 0.650, 0.720, 0.880, 1.100 and 1.500 s 27. The transverse (T2) relaxation rates of PhoBN were obtained with relaxation delays of 5.76, 17.28, 28.80, 40.32, 57.60, 69.12, 92.16, 138.24 and 172.80 ms

27

. The 1H-15N steady-state

heteronuclear NOE experiments were collected in an interleaved pulse sequences with and without proton saturation for 1 s

27

. The spin-relaxation data were measured for PhoBN at different

concentrations (0.30 mM and 0.87 mM). T1 and T2 values were calculated using the equations as described in reference 28. The hetero-NOE values were determined from the ratio of peak heights from experiments with and without 1H-saturation pulses. Rotational correlation time τc were given by Stokes’s

law

29

.

The

spectral

density

functions

were

analyzed

by

Relax

(http://www.nmr-relax.com/manual/) Concentration dilution experiments: Successive concentration dilution experiments were carried out with the concentrations of PhoBN at 1.42, 1.30, 1.20, 1.10, 1.00, 0.90, 0.80, 0.70, 0.60, 0.50, 0.40, 0.30 and 0.17 mM. Chemical shift perturbations (CSPs) were calculated using equation 1: ∆δ( 1 H N) 2 + ∆δ ( 15 N) 2

CSP =

(1)

where ∆δ (1HN) and ∆δ (15N) are the chemical shift differences between the backbone 1HN and

15

N

atoms at different concentrations. Dimerization constant K was calculated by fitting to the equation 2 30:

K=

[ A2 ] = f d [ A]2 2 [ A]0

f m2

(2)

where [A], [A2] and [A]0 are the concentrations of monomer and dimer, and total concentration, respectively. The quantities fm and fd are the mole fractions of protein A in the forms of monomer and



Page 8 of 31

dimer, respectively (fm+fd=1). CSP was used to calculate the dimerization constant K. The relationship between chemical shift perturbation and dimerization constant K is determined by equation 3.

∆δobsd = δm + fd(δd - δm) = δm + (δd - δm)

[1 + 8K[A] 0] 1 / 2 - 1 [1 + 8K[A]0] 1 / 2 + 1

(3)

where ∆δobsd is the observed chemical shift perturbations, which is the weighted average of changes between monomer and dimer. δm is the chemical shift of monomer, and δd is the chemical shift of dimer. Paramagnetic relaxation enhancement (PRE) experiments: Cysteine residue can be used to conjugate

with

the

thiospecific

spin-label

reagent

MTSL

((1-oxyl-2,

2,

5,

5-tetramethyl-3-pyrroline-3-methyl) methanethiosulfonate) purchased from Affymetrix-Anatrace. For site-specific spin labeling, four cysteine mutants of PhoBN, A114C, K110C, E89C and E23C, were prepared by mutation of PhoBN-C19A. These proteins were incubated with MTSL at 4℃ temperature for 24 hours with a molar ratio of 1:4. Excess MTSL was removed by desalting column and the conjugation was confirmed by MALDI-TOF mass spectrometry.

15

N-labeled PhoBN-C19A and

unlabeled PhoBN-C19A (A114C, K110C, E89C and E23C) which was conjugated with a MTSL probe were mixed together with a molar ratio of 1:1. All experiments were repeated after the spin labeling was reduced with 5-fold excess concentrated ascorbic acid to NMR samples. NMR samples were prepared by adding reduced agents at 25 ℃ and incubated for at least 2 hours before acquisition. Intermolecular PRE rates (Γ2) were obtained from the intensities of cross-peaks of backbone amide proton-nitrogen pairs in 15N-HSQC spectra of the paramagnetic and diamagnetic state (after addition of ascorbic acid). A two-time point measurement 31 provides a simple means of obtaining Γ2 rates which is the difference in transverse relaxation rates between the paramagnetic (R2, para) and diamagnetic (R2, dia)


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states of the macromolecule (equation 4).

Γ 2 = R2,para - R2,dia =

I (T )I (T ) 1 ln dia b para a Tb - Ta I dia (Ta )I para (Tb )

(4)

where Idia and Ipara are the peak intensities for diamagnetic and paramagnetic states recorded at two time points Ta and Tb (T=0 and ∆T), respectively.

Results and Discussion Backbone assignment of PhoBN indicates conformational inhomogeneity The isotopic labeled receiver domain PhoBN (1~125) was overexpressed and purified from E. coli. The HSQC spectrum of 15N-PhoBN shows relatively sharp and well-dispersed signals, but the peaks in the middle of the spectrum are broad and inhomogeneous (Figure 1a). We are able to assign 98 out of 117 non-proline residues. The unassigned residues are Glu11, Ala12, Ile14, Cys19, Glu23, Met55, Leu56, Gly58, Ile65, Lys66, Glu89, Asp90, Arg91, Val92, Phe107, Lys110, Glu111, Ile116, and Leu112. They are mainly located in loop β1-α1, loop β3-α3, helix α4 and helix α5. The inhomogeneity in peak intensities and signal broadening implies conformational exchanges in solution and does not facilitate the assignment. The secondary structure elements of PhoBN were predicted from chemical shifts using TALOS+. 13

Cα and 13Cβ chemical shifts were analyzed to indicate protein secondary structure in solution (Figure

1b). Even though only about 83.8% of the backbone assignments were accomplished, the chemical shift index (CSI) analysis suggests that PhoBN in solution adopts a conserved α/β global folding of response regulator. The secondary structure is almost the same as the crystal structure except a flexible N-terminus including β1 and α1. The assignment of helix α4 is not completed, indicating a much mobile conformation other than the rigid helix appeared in the crystal structure. In addition, it has been



Page 10 of 31

reported that helix α4 of the response regulator in OmpR/PhoB family displays remarkable different structures

32-34

. As for PhoBN, helix α4 is mostly exposed and connected to the active site.

Phosphorylation at Asp53 will lead to the movement of Thr83 and this can alter the N terminus of helix α4

35

. The orientation of α4 in PhoBN crystal structure is shifted along the helical axis upon

phosphorylation, and its preceding and following loops are rather flexible areas. The rest of the secondary structure of PhoBN, such as helix α2, α3, β4 and α5, is rigid and consistent with the crystal structure 36.

PhoBN forms dimer at high concentration 15

N relaxation parameters of backbone amides provide a powerful experimental approach for

detecting conformational dynamics of proteins at atomic resolution

37

. We measured the relaxation

parameters for the backbone amide nitrogens of PhoBN: 1H-15N nuclear Overhauser effect (NOE), rate constants for spin-lattice relaxation (R1), and spin-spin relaxation (R2) which are responsive to fast time scale (nanosecond to picosecond) motions. By comparing the relaxation rates of samples at different concentrations (0.30 mM and 0.87 mM), we found that R1 and R2 are both concentration-dependent, and R2 is fluctuating in a large range of values (Figure 2a). For most of the residues, the R2 values increase when the protein is at higher concentration. Based on R1 and R2 measurements, we calculated the overall rotational correlation times (τc) for PhoBN by Stoke’s law to evaluate the internal bond motions. The residues with heteronuclear NOEs less than 0.6 which indicate rapid local internal motions were excluded from the calculation. The correlation time τc is 13.79 ns for the protein at 0.30 mM, which is close to the expected value for monomeric PhoBN, whereas the τc measured at 0.87 mM is 16.22 ns that appears to be in exchange between monomeric and dimeric states.


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Due to monomer-to-dimer exchange or existence of possibly higher order oligomers, we did not use model-free analysis to obtain order parameter S2 values which are representative of overall amplitudes of fast scale dynamics. Instead, we used the simplest form of the Lipari-Szabo model-free spectral density function to derive a measure of the motion at particular frequencies (Figure 2b). J (ω) indicates the relative distribution of rotational diffusion motion frequency and describes the effect of intramolecular motion on relaxation. The main way to explain the dynamics of protein is reduced spectral density mapping J (0), J (ωN), and J (0.87ωH)

38

. These three spectral density functions are

related to the rapid motions of NH bonds. Smaller values of correspond to larger internal motions, while smaller values of indicate increasing , which are roughly anti-correlated with . As can be seen in Figure 2b, the smaller values of PhoBN at the concentration of 0.30 mM indicates larger internal motions in the monomeric state. In the case of , the values at 0.87 mM are obviously smaller than those at 0.30 mM, implying these residues are less mobile at higher concentration. For , the values for PhoBN are nearly superimposable at 0.30 mM and 0.87 mM, implying that they are independent of global tumbling. The analysis of reduced spectral density shows that several residues have obviously larger values at 0.87 mM than at 0.30 mM, such as Ile62, Q63, His67, R70, Arg93, Leu95, Ala114 and Ala118. The mapping shows they are located in helix α3 and α4-β5-α5 face (Figure 2b and Figure 5b) implying enhanced rigidity of these regions under the concentration of 0.87 mM. It means that the α4-β5-α5 face may be involved in the dimerization of apo PhoBN and influence the movements of these residues as the concentration increases.

The conformation of PhoBN dimer is not unique



Page 12 of 31

Dilution experiments were carried out in order to quantify the dimeric exchanges. 1H-15N HSQC spectra of PhoBN (Figure 3a) were recorded at a series of concentrations (from 1.40 mM to 0.17 mM). The expanded spectrum clearly illustrates the amides which have large chemical shift changes. The peak perturbations are not caused by the changes of pH or buffer viscosity as identified by the control experiment (supporting information Figure S1). The chemical shift perturbations of the residues larger than 15 Hz between 0.17 mM and 1.40 mM are shown in Figure 3b, and the dimerization constants (K) were calculated

30

. By taking the errors into account, the dimerization process of apo PhoBN is not

homogenous. By comparing the dimerization constants, we can generally classify them into at least two groups as listed in Table 1. When these perturbed residues are mapped on the crystal structure of apo PhoBN, it can be easily found that the residues with smaller dimerization constants, such as Tyr102, Ala114, and Ala118 are located on the α4-β5-α5 dimer interface as colored in blue in Figure 3c; the residues with larger constants, Glu16, Gln24 and Ser108, are on the α1-α5 dimer interface as colored in red in Figure 3c. It has been reported that apo PhoBN exhibits an equilibrium between different oligomeric states

19

. Our data agree with this conclusion and the mapping of perturbed residues

suggests that the dimer surface is not only restricted at the α1-α5 face

19

but also spreads among the

loops and external α4-β5-α5 face (Figure 3c). According to the values of dimerization constants, it is reasonable to assume that both interfaces are involved in the dimerization and the α1-α5 face is more preferable than the α4-β5-α5 face.

Dimerization interface analyzed by paramagnetic relaxation enhancement (PRE) To further characterize the dimerization orientation of apo PhoBN in solution, we used site-directed spin labeling with a nitroxide spin-label compound MTSL, covalently attached to a


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cysteine residue for intermolecular paramagnetic relaxation enhancement (PRE) experiments 39, which can be used to obtain intermolecular long distance (< 30 Å) information for lowly populated species 31, in our case, the transiently formed dimer of apo PhoBN. Wild-type PhoBN contains one cysteine residue at position 19, however, the trial on MTSL-linked Cys19 failed, because MTSL labeling at this position has a big impact on the protein structure (Supporting information Figure S2). Therefore, the single cysteine residue was introduced on a cysteine-less PhoBN-C19A mutant at position Ala114, Lys110, Glu89 and Glu23 respectively, to enable the conjugation of MTSL (Supporting information Figure S3 and S4). These residues were chosen because they are close to the expected dimer interface, and the MTSL-labeling is not likely to disrupt the protein conformation and dimerization which were confirmed by HSQC spectra (Supporting information Figure S5). The binding abilities of the mutants with the phosphoryl analog BeF3- were also confirmed by the 19F experiments (Supporting information Figure S6). The intermolecular PRE rates for amide protons were measured on an equimolar mixture of 15

N-labeled PhoBN C19A and unlabeled PhoBN C19A conjugated with a MTSL probe. The residues

with transverse PRE rates, Γ2, larger than 10 s-1 were mapped onto the crystal structure of apo PhoBN (1B00) in Figure 4. Basically, the spin labeling on the specifically introduced cysteine residue increases the relaxation rates of the nearby residues or those are spatially close to the spin. However, it can be clearly seen in Figure 4 that the PRE rates of a wide range of residues are perturbed by the paramagnetic probe. For instance, the spin labeling on A114C increased the relaxation rates of 27 amino acids beyond 22%. Some of them are located near A114C and the expected α1-α5 dimer interface, while the others are located at the helix α4, loop α4-β5 and strand β5 (Figure 4a). Unexpected perturbed residues can be also found in the cases of K110C and E89C, and E23C conjugation sites (Figure 4b, 4c and 4d).



In order to carefully examine the dimerization interfaces, the residues influenced by MTSL conjugated at K110C are illustrated and analyzed in Figure 5, in a color code of transverse PRE rate values, Γ2. The residues whose Γ2 are larger than 20 s-1 are colored in red and orange, and values between 10 ~ 20 s-1 are in yellow and blue. As can be seen in Figure 5a, most of the residues with larger PRE rates, such as Glu16, Gln24, Asn25, and Gly26, can be perfectly explained by the structure of apo PhoBN (1B00, Mode A) dimerized by the α1-α5 face. However, some residues such as Leu95, Ala99 and Tyr102 are far away from the paramagnetic center at K110C in a distance above 25 Å as indicated in Figure 5a. These residues can be well explained by the structure of BeF3--activated PhoBN (1ZES, Mode B) illustrated in Fig. 5b. In this model, Leu95 and Ala99 are very close to the intermolecular K110C, and Tyr102 is located within 14 Å through the α4-β5-α5 dimer interface (Figure 5b). Compared to the residues located near α1-α5 face, the PRE effects of Leu95, Ala99 and Tyr102 are much weaker, indicating that the active-like conformation of apo protein is less populated in solution than the dimer conformation in the active form, which is consistent with the results from the dilution experiments. Interestingly, the PRE effects of some residues, such as Glu33, Asp36, Trp54 and Ser60, which have large Γ2 values can not be explained by either of the dimerization mode in Figure 5a or 5b. For instance, the backbone amide nitrogen of Asp36 is 27.0 Å and 31.2 Å away from that of the intermolecular K110C, respectively, implying some unexpected dimerization conformations in solution. In the crystal structure of BeF3--activated PhoBN (1ZES), there are three PhoBN molecules in an asymmetric unit, two of them forming a symmetric α4-β5-α5 dimer, the third forming a dimer with one of the symmetry-related molecule using the α1 and α2 interface. The latter conformation has been illustrated as Mode C in Figure 5c, where Glu33 and Asp36 are close to the intermolecular K110C.


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However, Mode C can barely explain the large Γ2 effects of Trp54 and Ser60, who are in distances around 22 Å to K110C (Figure 5c and 5d), implying that there must be some other dimer conformations in solution. Therefore, all the results agree that apo PhoBN forms more than one dimer interfaces in solution and the protein has a variety of conformational exchanges.

Conclusions Dimerization of response regulator has been considered to be a necessary process in OmpR/PhoB family for bacterial signal transduction11,40,41. The dimerization using the α4-β5-α5 face has been widely observed in many receiver domains in their apo or active states, such as PhoP 42, ArcA 43 and TorRN

41

from E.coli. Although the physiological importance of the α1-α5 face has not been

determined for PhoBN, in the case of DesR, the α1-α5 face plays an important role in holo state 44. We found that, in addition to the monomer-to-dimer exchange, the inactive PhoBN can form different dimer arrangements which are independent of phosphorylation. Detailed data analyses from concentration dilution experiments and PRE measurements show multiple dimer interfaces undergoing conformational exchanges. The equilibrium of the dimer arrangements is not the same as the equilibrium between active and inactive forms, because each monomer unit in the PhoBN dimer shows conformation of inactive state as indicated by HSQC spectra and CSI prediction. It has been widely accepted that the transient protein-protein interaction plays an important role in signal transductions. Phosphorylation might stabilize a low-abundant preexisting conformation in the nonphosphorylated population. The transient homodimer interaction could also be a trigger associated with the phosphorylation process of apo PhoBN. For example, Tyr102 was demonstrated to be involved in the dimerization process from the data of concentration dilution and PRE experiments (Figure 3c and



Figure 5b). In the crystal structures of apo PhoBN and BeF3--activated PhoBN, the sidechain of Tyr102 shows large conformational changes upon phosphorylation. The re-orientation of tyrosine sidechain accompanied with the nearby threonine is called ‘Y-T coupling’ mechanism which is important for the biological functions

45

. The perturbation of Tyr102 of apo PhoBN suggests that there are transient

interactions during the dimerization, and the observation of low populated α4-β5-α5 dimer in apo state may be a general mechanism for inducing structural changes leading to the activation of PhoBN. In summary, our results revealed that apo PhoBN forms a dimer in solution using more than one dimer interfaces. Our findings imply that the switch of dimer interfaces which is independent of phosphorylation may help to form active states of PhoBN in solution. The transient homodimer interactions of apo PhoBN might be relative to its biological function and could provide useful information to understand the diverse signal transduction mechanism of response regulators.

Conflict of interest statement All authors declare no conflict of interest.

Acknowledgments This work was supported by the grants from National Key R&D Program of China (#2017YFA0505400) and the National Science Foundation of China (#21573280 and #21603268).

Supporting Information Available: The overlay of 2D 1H-15N HSQC spectra of wt-PhoBN in the absence and presence of RR468 (Figure S1). The 1H-15N HSQC spectra of wt-PhoBN with and without MTSL (Figure S2). The overlay of 2D 1H-15N HSQC spectra of wt-PhoBN and PhoBN-C19A (Figure


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S3). The overlay of 2D 1H-15N HSQC spectra of PhoBN-C19A and four mutants prepared for the PRE experiments (Figure S4). The overlay of 1H-15N HSQC spectra of PhoBN mutants with and without MTSL (Figure S5). The 19F NMR spectra (600 MHz, 25℃) of PhoBN and its mutants in the presence of BeF3- (Figure S6).

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Table 1. Dimerization constant (K) of apo PhoBN calculated from concentration dilution experiments. Residue No. K (mM-1) error (mM-1)

H67

G86

Y102

A114

A118

G61

E16

Q24

E87

S108

0.45

0.64

0.38

0.37

0.22

1.26

3.06

2.91

3.33

2.95

±0.38

±0.45

±0.21

±0.11

±0.11

±2.21

±1.86

±1.38

±3.03

±1.91



Figure Legends

Figure 1 2D 1H-15N HSQC spectrum and secondary structure analysis of PhoBN. (a) Backbone resonance assignments of PhoBN are labeled by one-letter amino acid code and sequence number. Regions with cross-peaks partially overlapped are expanded. (b) Secondary structure elements were predicted using TALOS+. Chemical shift differences between ∆δCα and ∆δCβ were used to indicate the secondary structure.

Figure 2 Backbone dynamics of apo PhoBN. (a) 1H-15N heteronuclear NOEs, relaxation rates R1 and R2 as a function of residue number. (b) Spectral density functions at three different frequencies (blue: 0.30 mM; red: 0.87 mM). The residues with larger values at 0.87 mM and related to dimer interface are indicated in black boxes.

Figure 3 Concentration dilution experiments of apo PhoBN. (a) 1H-15N HSQC spectra of apo PhoBN at different concentrations (blue: 1.00 mM, red: 0.60 mM, green: 0.30 mM, purple: 0.17 mM). The representative region which has chemical shift perturbations is enlarged in the box. (b) Changes of chemical shift values over protein concentrations for all perturbed residues. The chemical shift differences are expressed as (δH+δN)1/2, in which δH and δN are in Hz. (c) Cartoon representative of apo PhoBN (PDB: 1B00). Residues which have large chemical shift changes are colored in red (on the α1-α5 dimer interface) and blue (away from the α1-α5 dimer interface). The most perturbed residues have been indicated by letters.


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Figure 4 Intermolecular 1H transverse PRE Γ2 rates measured on an equimolar mixture (0.3 mM each) of

15

N-labeled PhoBN mutant (C19A) and unlabeled PhoBN C19A (A114C, K110C, E89C and E23C)

conjugated with a MTSL probe. Inserts: residues with observed PRE values > 10 s-1 are mapped on the crystal structure of apo PhoBN (PDB: 1B00) in red. Spin-labeling sites are colored purple.

Figure 5 Locations of the residues having large PRE effect from K110C-MTSL conjugation in different dimerization modes. The MTSL conjugated K110C is shown as red sphere. Different PRE-effect ranges are displayed in different color codes respectively (blue: 10 ~ 15 s-1; yellow: 15 ~ 20 s-1; orange: 20 ~ 30 s-1; red: > 30 s-1). The distances between the residues and the paramagnetic probe are marked by brown dotted lines. (a) Inactive PhoBN dimerization by using α1-α5 face (Mode A, PDB:1B00); (b) Active PhoBN dimerization by using α4-β5-α5 face (Mode B, PDB:1ZES); (c) Active PhoBN dimerization in other way (Mode C, PDB:1ZES). (d) Classification statistics of the different PRE effect ranges. Residues highlighted in red are shown in Mode C.



TOC Graphic


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2D [1H-15N]-HSQC spectrum and secondary structure analysis of PhoBN. 118x166mm (300 x 300 DPI)



Backbone dynamics of apo PhoBN 169x174mm (300 x 300 DPI)


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Concentration dilution experiments of apo PhoBN 144x130mm (300 x 300 DPI)



Intermolecular 1H transverse PRE Γ2 rates measured on an equimolar mixture (0.3 mM each) of 15N-labeled PhoBN mutant (C19A) and unlabeled PhoBN C19A (A114C, K110C, E89C and E23C) conjugated with a MTSL probe 170x141mm (300 x 300 DPI)


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Locations of the residues having large PRE effect from K110C-MTSL conjugation in different dimerization modes 162x141mm (300 x 300 DPI)


Dimerization and Conformational Exchanges of the ... - ACS Publications

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