
==== Front
Proc Natl Acad Sci U S A
Proc Natl Acad Sci U S A
PNAS
Proceedings of the National Academy of Sciences of the United States of America
0027-8424
1091-6490
National Academy of Sciences

39190358
202404472
10.1073/pnas.2404472121
datasetDatasetresearch-articleResearch Articlebiophys-bioBiophysics and Computational Biology408
Biological Sciences
Biophysics and Computational Biology
Direct evidence for a deprotonated lysine serving as a H-bond “acceptor” in a photoreceptor protein
Nagae Takayuki a https://orcid.org/0000-0001-7016-5183

Takeda Mitsuhiro a
Noji Tomoyasu b c https://orcid.org/0000-0001-9468-2038

Saito Keisuke b c https://orcid.org/0000-0002-2293-9743

Aoyama Hiroshi a
Miyanoiri Yohei d https://orcid.org/0000-0001-6889-5160

Ito Yutaka e https://orcid.org/0000-0002-1030-4660

Kainosho Masatsune e https://orcid.org/0000-0002-5229-7251

Hirose Yuu f https://orcid.org/0000-0003-1116-8979

Ishikita Hiroshi hiro@appchem.t.u-tokyo.ac.jp
b c 1 https://orcid.org/0000-0002-5849-8150

Mishima Masaki mmisima@toyaku.ac.jp
a 1 https://orcid.org/0000-0001-7626-7287

aDepartment of Molecular Biophysics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan
bResearch Center for Advanced Science and Technology, The University of Tokyo, Meguro-ku, Tokyo 153-8904, Japan
cDepartment of Applied Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 118-8656, Japan
dResearch Center for State-of-the-Art Functional Protein Analysis, Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan
eDepartment of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Hachioji 192-0397, Japan
fDepartment of Applied Chemistry and Life Science, Toyohashi University of Technology, Toyohashi, Aichi 441-8580, Japan
1To whom correspondence may be addressed. Email: hiro@appchem.t.u-tokyo.ac.jp or mmisima@toyaku.ac.jp.
Edited by Adriaan Bax, NIH, Bethesda, MD; received March 22, 2024; accepted July 11, 2024

27 8 2024
3 9 2024
27 8 2024
121 36 e240447212122 3 2024
11 7 2024
Copyright © 2024 the Author(s). Published by PNAS.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

Significance

H-bonds play important roles in protein structure and function. Understanding H-bonds is essential for insights into biological processes. Here, we report the identification of a “deprotonated charge-neutral (NH2 form)” sidechain of lysine in the photosensor protein RcaE. Notably, it serves as a H-bond “acceptor” as revealed by NMR via trans-H-bond J coupling. Of the two RcaE photostates, namely the red-light-absorbing (Pr) state and green-light-absorbing (Pg) state, the NH2 form of the lysine sidechain is only present in the Pg state. In the Pg state, the lysine sidechain acts as a H-bond acceptor whereas it acts as a H-bond donor in the Pr state. Thus, the lysine acts as both donor and acceptor, switching between states upon light absorption.

Deprotonation or suppression of the pKa of the amino group of a lysine sidechain is a widely recognized phenomenon whereby the sidechain amino group transiently can act as a nucleophile at the active site of enzymatic reactions. However, a deprotonated lysine and its molecular interactions have not been directly experimentally detected. Here, we demonstrate a deprotonated lysine stably serving as an “acceptor” in a H-bond between the photosensor protein RcaE and its chromophore. Signal splitting and trans-H-bond J coupling observed by NMR spectroscopy provide direct evidence that Lys261 is deprotonated and serves as a H-bond acceptor for the chromophore NH group. Quantum mechanical/molecular mechanical calculations also indicate that this H-bond exists stably. Interestingly, the sidechain amino group of the lysine can act as both donor and acceptor. The remarkable shift in the H-bond characteristics arises from a decrease in solvation, triggered by photoisomerization. Our results provide insights into the dual role of this lysine. This mechanism has broad implications for other biological reactions in which lysine plays a role.

NMR
H-bond
lysine
trans-H-bond J coupling
cyanobacteriochrome
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pmcH-bonds play a crucial role in protein structure and function. Due to their lower bond energies compared to covalent bonds, H-bonds are involved in essential processes such as protein–ligand association, dissociation, and proton transfer, all of which facilitate efficient catalytic activity of enzymes. H-bonds form between donor and acceptor moieties, where the moiety with a higher pKa value acts as a H-bond donor (e.g., basic groups), and that with a lower pKa value acts as a H-bond acceptor (e.g., acidic groups) (1). In protein environments, lysine and arginine are often protonated and positively charged, making them H-bond donors. The presence of a “deprotonated arginine” (Arg52) was speculated, based on the interpretation of neutron and X-ray crystal structures in water-soluble photoactive yellow protein (PYP), regardless of its proximity to the protein bulk surface (2). Originally, the concept of this deprotonated arginine was introduced as “experimental evidence” to support the existence of a low-barrier H-bond near the residue (2). However, theoretical studies suggested that the arginine residue is protonated in the crystal (3), and this was further supported by solution NMR studies (4). Therefore, there is little basis for the presence of a deprotonated arginine in PYP. In contrast, the transient deprotonation or suppression of the pKa of lysine sidechains is a widely recognized phenomenon. The presence of a “deprotonated lysine” (Lys115) was first reported in acetoacetate decarboxylase (AADase) (5, 6). Initially, it was speculated, based on the amino acid sequence, that the decrease in pKa of Lys115 (to ~6) was due to the presence of Lys116 (5). However, further analysis of the crystal structure revealed that the two sidechains are oriented in opposite directions, excluding the contribution of electrostatic repulsion to the decrease in pKa (Lys115) (7). Instead, loss of solvation at the Lys115 moiety is predominantly responsible for its low pKa value (8). This highlights Lys115 in AADase as an example demonstrating the existence of a deprotonated basic residue in a protein environment and underscores its significance in the catalytic activity. Similarly, in Exiguobacterium sibiricum rhodopsin, Lys96, the primary proton uptake residue for the retinal Schiff base, remains deprotonated in the ground state structure owing to the loss of solvation in the protein environment (9). A transient deprotonation or suppression of the pKa of a lysine sidechain has been suggested in E2 Ubc9 conjugating proteins (10, 11), histone lysine methyltransferase (12), and Ser-Lys dyad enzymes (13). However, these reports are based on indirect evidence such as activity measurements.

RcaE is a cyanobacteriochrome (CBCR) and serves as a photosensor in cyanobacteria. For photoperception, RcaE utilizes phycocyanobilin (PCB), a tetrapyrrole chromophore that is bound to the cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) domain, and undergoes reversible photoconversion between two distinct light-absorbing states triggered by C15-Z/C15-E photoisomerization of a PCB. RcaE adopts red-light-absorbing (Pr) and green-light-absorbing (Pg) states (14). To better understand the mechanism of photoconversion of RcaE, we previously reported the crystal structures of the GAF domain in the Pr state (15) and Pg state (16). Here, we report the identification of a “charge-neutral (NH2 form)” sidechain of lysine in the Pg state as revealed by NMR. Notably, the NH2 group of the sidechain becomes a H-bond “acceptor.”

Results

Protonation of the N Atom of the D-Ring.

Inspection of the X-ray crystal structure of the GAF domain of RcaE in the Pg state (PDB:8K9O) revealed a close contact between the N atom of the D-ring in PCB and the Nζ atom of the sidechain of Lys261 (the N…N distance = 3.07 Å) (Fig. 1A) (16), which is in contrast to the Pr state structure (PDB:7CKV) (the N…N distance >10 Å) (15). This close contact was investigated, using NMR spectroscopy. By comparing the NMR spectra of RcaE holoproteins which consist of unlabeled apoprotein with PCB selectively 13C labeled in three different patterns (labeling pattern I: 1,3,4,5,6,8,9,10,11,12,14,15,16,18,19-13C, labeling pattern II: 4,5,9,10,11,15,19-13C, and labeling pattern III: 1,3,6,8,12,14,16,18-13C; see SI Appendix, Fig. S1) with uniform 15N labeling, based on the previously established method by Rockwell et al. (17), an NH signal in 1H-15N heteronuclear single-quantum coherence (HSQC) was unambiguously assigned as the protonated N atom (NH group) of the D-ring of PCB (SI Appendix, Fig. S1 and Supplemental Text). We next monitored a one-bond isotope shift of the N atom of the D-ring in the Pg state caused by 2H addition. A one-bond isotope shift is known to be useful in judging whether an N atom contributes to a H-bond, and whether it acts as a donor (N-H) or an acceptor (N) in an N–H…N type H-bond (18). We performed 1D 15N NMR experiments with various 2H2O concentration solvents (0%, 50%, and 100% 2H2O). Up-field one-bond isotope shift (~0.86 ppm) upon binding of 2H to the N atom of the D-ring was clearly observed (Fig. 1B), indicating that the N-H of the D-ring contributes to formation of a H-bond, and that the N atom acts as a donor. Fig. 1B shows that the chemical exchange between N-H and N-D proceeds in a slow-exchange manner on the NMR time scale. We estimated that the exchange rate is slower than ~40 Hz by comparing simulated spectra (SI Appendix, Fig. S2). This also supports the proposal that the proton is involved in a H-bond. Thus, it seems highly likely that closely contacted Lys261 sidechain acts as an acceptor in the H-bond with the NH group of the D-ring (Fig. 1C).

Fig. 1. Lys261 interacts with the D-ring in the Pg state. (A) Close-up view of the crystal structure of the GAF domain of RcaE in the Pg state. Possible H-bonds are shown as dotted lines. Interaction between the sidechain N atom of Lys261 and the N atom of the D-ring in PCB is colored in red, and the distance is also shown in Å. Red spheres represent oxygen atoms of water molecules. (B) Isotope shift of the N atom of the D-ring in PCB caused by deuterium (2H). The superposition of 15N 1D spectra of RcaE holoprotein (unlabeled apoprotein with 15N labeled PCB) is shown. In these spectra, only 15N signals from PCB are detected. Green, red, and blue indicate the 2H2O content of the buffers, 0, 50, and 100%, respectively. (C) Schematic representation of the proposed H-bond between the sidechain NH2 of Lys261 and the N atom of the D-ring of PCB. The H-bond is shown as a dotted red line.

Lys261 Protonation State Deduced from an Anomalous Chemical Shift.

To validate the interpretation that Lys261 acts as an acceptor, we next clarified the protonation state of the amino group of the Lys261 sidechain. A 1H-15N HSQC experiment covering a wide-range on the 15N axis was conducted. In the Pg state, the 1H-15N HSQC spectrum exhibits an anomalous signal with chemical shifts of 0.69 ppm for 1H and 27.4 ppm for 15N (Fig. 2A). These chemical shifts correspond to extremely high fields, which are in contrast to the chemical shifts of ~8 ppm for 1H and ~120 ppm for 15N observed with typical backbone NH groups. Notably, a lysine sidechain introduced into the hydrophobic interior of the V66K mutant of the Δ+PHS SNase (44 to 49 deleted hyperstable form of Staphylococcus nuclease) (19) exhibited comparable chemical shifts of 0.81 for 1H and 23.3 ppm for 15N (20). The amino group of the lysine is known to adopt an NH2 form (20). The similarity in chemical shifts between V66K Δ+PHS SNase and Pg state RcaE strongly suggests that the anomalous signal in the Pg state can most likely be attributed to the presence of a NζH2 moiety of the lysine sidechain. Importantly, the absence of the corresponding signal in the Pr state (SI Appendix, Fig. S3A) indicates the absence of deprotonated lysine, which is consistent with the lack of a H-bond between the D-ring of PCB and Lys261 in the Pr state structure (SI Appendix, Fig. S3B).

Fig. 2. Identification of Lys261-NζH2. (A) 1H-15N HSQC spectrum of the Pg form covering a wide-range on the 15N axis. 15N labeled RcaE holoprotein (uniformly 15N labeled apoprotein with 15N labeled PCB) was used. (B) 1H-15N HISQC spectrum of the Pg form. 15N labeled RcaE holoprotein (uniformly 15N labeled apoprotein with 15N labeled PCB) was used (C) 13C 1D NMR spectra of Pg form. The ε-postion-13C labeled lysine is incorporated into unlabeled RcaE holoprotein. The 2H2O content of the buffer was either 0, 50, or 100%.

Lys261 Protonation State Revealed by Signal Splitting and Two-Bond Isotope Shift.

In addition to the anomalous chemical shift, we investigated the protonation state using more direct methods. A 1H coupled 1H-15N heteronuclear in-phase single-quantum correlation (HISQC) experiment for 15N PCB and 15N lysine labeled RcaE in the Pg state was conducted. The signal is split as a 1:2:1 triplet (Fig. 2B), which clearly indicates that the anomalous signal originates from the –NH2 moiety. We also investigated the effect of deuteration on the isotope shifts of 13C at the ε positions adjacent to [Nζ2H3/Nζ2H2] of the lysine sidechains (SI Appendix, Fig. S4A). The magnitude of the isotope shift of Lys261 (0.21 ppm) is considerably smaller than that of the other lysine residues, namely Lys165, Lys196, and Lys259 (~0.27 ppm) (Fig. 2C and SI Appendix, Fig. S4B). This finding strongly supports the conclusion that Lys261 adopts the deprotonated NH2 form, whereas the other lysine residues adopt the protonated +NH3 form. These observed secondary isotope shifts are consistent with those reported for V66K Δ+PHS SNase (21).

Deprotonated Lys261 Sidechain Serving as a H-Bond Acceptor Revealed by Trans-H-Bond J Coupling.

Using a HNN correlation spectroscopy (HNN-COSY) pulse sequence originally designed for detection of H-bonds in DNA base pairs (22, 23), the H-bond between the D-ring NH of PCB and the Nζ of Lys261 can be detected via h2JNN coupling. In this context, hnJAB denotes a trans-H-bond scalar coupling between nuclei A and B over n chemical bonds (e.g., n = 2 for the N–H…N bond with N-H and H…N, Fig. 3A). To obtain the correlation between the protein part and the PCB, we used uniformly 15N labeled apoprotein and 15N labeled PCB. The observed cross-peak (Fig. 3A) indicates transfer of excited magnetization from the 1H of the D-ring NH via the 15N of the D-ring NH to the 15N of Lys261 Nζ, and eventually back to the 1H of the D-ring NH. This observation of the magnetization transfer serves as direct evidence for the existence of a H-bond between the D-ring NH (donor) and the Lys261 Nζ (acceptor). The magnitude of the h2JNN coupling is roughly estimated to be 3 Hz based on the intensity ratio of the cross- and diagonal peaks. Using 1H-decoupled 1D 1H-15N insensitive nuclei enhanced by polarization transfer (INEPT) NMR measurements, we investigated whether signal splitting of the Nζ of Lys261 sidechain caused by h2JNN could be observed. The results showed that apparent splitting was not observed. However, signal broadening was observed implying that the magnitude of the coupling is small (SI Appendix, Fig. S5A). The broadening is eliminated by 15N homonuclear long-observation-window band-selective homonuclear decoupling (LOW-BASHD) (24) applied to 134 ppm corresponding to the chemical shift of the ND of the PCB (SI Appendix, Fig. S5A), which is consistent with the existence of J coupling between the Nζ of the Lys261 sidechain and ND of the PCB. Furthermore, by comparing with a series of simulated spectra made by applying various magnitudes of J coupling, the magnitude is estimated to be around 3 Hz (SI Appendix, Fig. S5B). Finally, the value was quantified by a 1D 1H-15N spin-echo difference transverse relaxation-optimized spectroscopy (TROSY) pulse sequence (SI Appendix, Fig. S6) (25, 26). To minimize signal overlap, which could interfere with quantification, we performed selective 15N lysine labeling in addition to 15N PCB labeling. The intensity of the D-ring-NH signal decreases due to the h2JNN coupling as the dephasing period increases (Fig. 3B). The coupling is pronounced upon irradiation of a selective 180° pulse along the 15N axis at 28 ppm, which corresponds to the chemical shift of 15N in the Lys261 Nζ. The magnitude of the coupling constant is determined to be 2.5 ± 0.1 Hz (Fig. 3C and SI Appendix, Supplemental Text), which is in line with the previously reported magnitude of 2 to 5 Hz for typical H-bonds between backbone NH groups (donor) and histidine sidechain N atoms (acceptor) (26, 27).

Fig. 3. Observation h2JNN of H-bond (D-ring NH…NζH2-Lys261). (A) HNN-COSY spectrum of the Pg form. 15N labeled RcaE holoprotein (uniformly 15N labeled apoprotein with 15N labeled PCB) was used. The cross-peak arising via h2JNN is indicated by the arrow. Black and red contours represent positive and negative signals. The connectivity giving h2JNN is shown in the box. (B)1H-15N spin-echo difference TROSY spectra of the Pg form. For the measurements, 15N-Lys-15N PCB RcaE holoprotein (unlabeled apoprotein incorporating 15N labeled lysine with 15N labeled PCB) was used. Black, red, green, blue, pink, cyan, brown, and navy indicate dephasing delays of 0, 20, 40, 60, 80, 100, 120, and 140 ms, respectively. (C) Amplitude modulation of the NH signal of the D-ring. h2JNN modulation curve fitting for normalized amplitude of the NH signal of NH of the D-ring in the 1D 1H-15N spin-echo difference TROSY. The data plotted were normalized by setting the initial intensity equal to 1 to facilitate the analyses. Data were fitted with the equation I(2τNN) = A cos(π · h2JNN · 2τNN) to obtain A and J, where 2τNN is 0, 20, 40, 60, 80, 100, 120, and 140 ms. The h2JNN fitting is shown as a solid line, and the fitted h2JNN value is listed on the side with the error.

Theoretical Calculations.

To investigate the energetics of deprotonated Lys261, we performed quantum mechanical/molecular mechanical (QM/MM) calculations using the Pg crystal structure (PDB:8K9O) taking into consideration the protein environment. When Lys261 is protonated, the NH group of the D-ring experiences significant instability, leading to its displacement out of the D-ring plane (Fig. 4A). In contrast, deprotonated Lys261 accepts a H-bond from the NH group of the D-ring (Fig. 4B). The protonation state of the propionic group, which does not form a H-bond with the protein, did not affect the results (SI Appendix, Table S1). The potential-energy profile for the H-bond between deprotonated Lys261 and the NH group of the D-ring indicates that the proton is predominantly localized at the D-ring moiety. That is, the pKa value of the D-ring is higher than the pKa value of Lys261 in the Pg protein environment (Fig. 4C). Consequently, Lys261 remains deprotonated in the QM/MM-optimized structure, regardless of the initial H atom position along the H-bond. Fig. 4C indicates that there is a high activation barrier, consistent with the slow-exchange rate of the D-ring NH shown in the H-D exchange experiment (Fig. 1B). Accordingly, rotation of the H-bond around the Cε-Nζ bond of Lys261 should be very slow. Importantly, the chemical shift of Lys261-15 Nζ in the NH2 state obtained in QM/MM calculations (26.4 ppm), corresponds well to the experimental value of 27.4 ppm. In contrast, the calculated chemical shift of the +NH3 state is 34.3 ppm, clearly distinct from the calculated and measured chemical shifts of the NH2 state. Additionally, based on the QM/MM structure in the Pg state, in which the distance between ND and Nζ is 2.95 Å and the angle ∠NDHNζ is 169.7° (calculated using the large QM region with deprotonated Lys261 and deprotonated propionic groups of the PCB), the trans-H-bond J coupling h2JNN was calculated to be 4.6 Hz. The magnitude is slightly larger than the experimental value of 2.5 Hz. The discrepancy is probably due to reduction of the experimental value by some exchange phenomena. Furthermore, we examined the influence of the protonation state of Lys261 on the absorption wavelength of the PCB chromophore. The absorption wavelength of the PCB chromophore calculated in the presence of deprotonated Lys261 (510 nm) is closer to the experimentally measured absorption wavelength (532 nm) than that calculated in the presence of protonated Lys261 (594 nm) (SI Appendix, Table S2).

Fig. 4. QM/MM calculations using the Pg structure. (A and B) The PCB (gray) and Lys261(magenta) are shown as a stick model. The hydrogen atoms of Hζ of the Lys261 sidechain and the NH group of the D-ring are shown as cyan balls. The distances between the Nζ of Lys261 sidechain and the ND of the PCB are shown as red dotted lines, and the values are indicated in Å. (A) QM/MM-optimized geometry with protonated Lys261. (B) QM/MM-optimized geometry with deprotonated Lys261. (C) Potential-energy profile for the H-bond between deprotonated Lys261 and the NH group of the D-ring in the Pg protein environment.

Discussion

All of the observations described above are consistent with the idea that deprotonated Lys261 forms a H-bond with the NH group of the D-ring in the Pg crystal structure, thus serving as a H-bond acceptor. The distribution pattern of water molecules analyzed using a three-dimensional reference interaction site model (28) demonstrates the significant hydrophobic nature of the protein environment near Lys261 in the Pg crystal structure compared to the Pr crystal structure (SI Appendix, Fig. S7A). The result is consistent with the presence of a water molecule near Lys261 in the Pr crystal structure and its absence in the Pg crystal structure. These observations suggest that deprotonation of Lys261 can be attributed to the loss of solvation in the Pg structure, a deprotonation mechanism similar to that reported for AADase (8). Since there are no counterions such as COO− and no solvent molecules around the Lys261 sidechain in the hydrophobic environment in the Pg state, ionization of Lys261 sidechain is not thermodynamically favored in terms of its Born energy. This is the possible driving force for the formation of the neutral state of the Lys261 sidechain.

Overall, the results presented here highlight the dual role of Lys261 in RcaE. This lysine residue serves as a H-bond “donor” to the propionic group of PCB (PCB-COO−…+NζH3-Lys261) in the Pr state, whereas it serves as a H-bond acceptor for the D-ring NH group of PCB (D-ring NH…NζH2-Lys261) in the Pg state (Fig. 5). Thus, the sidechain amino group of the lysine can play both the roles of donor and acceptor via changing its pKa. The remarkable pKa shift arises from a decrease in solvation at the Lys261 moiety, triggered by the photoisomerization of the C=C bond linking the C and D-rings, along with subsequent protein conformational changes. Exclusion of water molecules in the Pg state is caused by structural changes which close the “leaky bucket” structure made by a loop structure found in the Pr state (15) (SI Appendix, Fig. S7B). In the Pg state, the loop changes to a β-strand forming a part of a layer of a β-sheet (SI Appendix, Fig. S7B). In the Pg state crystal structure (PDB:8K9O), Leu249 is located in the vicinity of the PCB and contributes to the hydrophobic environment. A L249H mutation showed destabilization of the Pg state (14), which demonstrates the importance of the hydrophobicity. Notably, the K261M mutation had little effect on the Pg state formation, but it exhibited a severe effect on the Pr state formation (14). This methionine (K261M) may give rise to a hydrophobic interaction with the PCB instead of the H-bond in which Lys261 acts as an acceptor (D-ring NH…NζH2-Lys261). However, it cannot serve as an alternative to the H-bond with Lys261 as a donor (PCB-COO−…+NζH3-Lys261) which is indispensable for stabilizing the Pr structure. Accordingly, we presume that the employment of a lysine residue at the 261st position allows for both the Pg and Pr formations, since lysine is a two-way player (both a donor and an acceptor) for H-bond formation. In fact, the lysine residue is conserved throughout the green/red subfamily of CBCR (14).

Fig. 5. Schematic drawing of deprotonation/protonation of Lys261 along with photoconversion. Deprotonated sidechain of Lys261 serving as a H-bond acceptor in the Pg form (Left) (PDB:8K9O). Protonated sidechain of Lys261 serving as a H-bond (salt bridge, bifurcated) donor in the Pr form (Left) (PDB:7CKV). Oxygen atoms of water molecules are shown as red spheres. Gray and green meshes represent electron density and difference electron density maps are contoured at 1.0 σ and 3.0 σ, respectively.

In conclusion, we have identified a “charge-neutral (NH2 form)” sidechain of lysine in a photosensor protein, which acts as a H-bond acceptor. The fully buried lysine of V66K Δ+PHS SNase makes no H-bonds and to our knowledge, a H-bond made by lysine where it acts as an acceptor is unprecedented. This is in sharp contrast to the common belief in biochemistry, in which the lysine sidechain is believed to adopt a charged form (+NH3), and act as a H-bond donor. The knowledge provided by our findings also demonstrates that there is a need to reconsider the existence of cryptic interactions formed by lysine, since structural biology is based on coordinates from X-ray crystallography which lack information about hydrogen atoms (protons).

Materials and Methods

Expression and Purification of RcaE Holoprotein Containing 15N,13C-Selectively Labeled PCB, and RcaE Holoprotein Containing 15N-Lys and 15N Labeled PCB.

Protein samples were prepared in essentially the same way as previously described (15). Briefly, the GAF domain (residues 164 to 313) of RcaE was cloned into plasmid pET-28 (Novagen). The construct was expressed in Escherichia coli strain BL21(DE3) star (Invitrogen) as a fusion protein with a His tag, together with PCB biosynthetic plasmid pKT271 (29). The harvested cells were lysed via sonication. The His-tagged protein was purified using a column of Ni-Sepharose 4B (Qiagen), followed by gel filtration using a Superdex 75 column (Cytiva) equilibrated in 50 mM HEPES-NaOH (pH 7.5) buffer containing 50 mM KCl. The purified sample was stored in buffer containing 50 mM HEPES-NaOH (pH 7.5) and 50 mM KCl.

For preparation of unlabeled RcaE holoprotein containing 15N,13C-selectively labeled PCB, selectively labeled 5-aminolevulinic acid (5-ALA), a metabolic precursor of PCB was added to M9 medium 0.5 h before induction. [4,5-13C, 15N] 5-ALA, [4-13C, 15N] 5-ALA and [5-13C, 15N] 5-ALA were used to produce 1,3,4,5,6,8,9,10,11,12,14,15,16,18-13C labeled PCB, 1,3,6,8,12,14,16,18-13C labeled PCB, and 4,5,9,10,11,15,19-13C labeled PCB, respectively (SI Appendix, Fig. S1). The partial PCB labeling was conducted with reference to literature by Rockwell et al. (17). These selectively labeled 5-ALAs were synthesized from [U-13C2, 15N] glycine, [1-13C, 15N] glycine, and [2-13C2, 15N] glycine (30) (SI Appendix, Fig. S1). For preparation of Lys specific 15N labeled RcaE holoproteins with 15N labeled PCB, 15N labeled Lys and 5-ALA were added to M9 medium 0.5 h before induction. The 13C,15N labeled RcaE holoprotein for standard triple resonance NMR measurements was expressed and purified in the same manner using 15NH4Cl and 13C6 glucose.

NMR Spectroscopy.

Prior to all NMR measurements, the NMR samples were irradiated with green or red light by light-emitting diode (LED). All NMR spectra were acquired under dark conditions, and they were processed and analyzed using Topspin 3.62 (Bruker). The chemical shifts for 1H, 13C, and 15N were primarily referenced to the methyl proton signal of the internal DSS according to the IUPAC recommendation (31). Using standard heteronuclear NMR techniques (32), we assigned resonances for almost all the protein (1H, 15N, and 13C for protein backbone and sidechain) including the anomalous NH signal (Lys261).

1H-Decoupled 1D-15N Measurements for Isotope Shift Monitoring.

1 mM 15N PCB labeled RcaE holoprotein (unlabeled apoprotein with 15N labeled PCB) was dissolved in buffer containing 10 mM Tris-HCl, pH 7.5, 50 mM KCl, and 0, 50, or 100% D2O (2H2O).  A 4.1 mm o.d. Shigemi tube containing the RcaE solution was inserted into a 5 mm o.d. outer tube containing D2O for the 2H lock signal. The measurements were performed on the Pg state.

1H-15N HSQC and 1H-(15N)-13C HNC.

The final concentrations of RcaE holoproteins (unlabeled apoprotein with selectively 13C and uniformly 15N labeled PCB) were ~200 μM. NMR samples were dissolved in buffer containing 10 mM Tris-HCl, pH 7.5, 50 mM KCl, and 7% D2O. Spectra were recorded at 600 MHz at 1H frequency at 298 K on a Bruker AVANCE 600 equipped with a TXI probe. The 1H-(15N)-13C HNC was conducted with the essentially identical pulse sequence with widely used HNCO.

1H-15N HSQC Covering Wide-Range 15N, 1H Coupled 1H-15N HISQC and HNN-COSY.

The final concentration of 15N labeled RcaE holoprotein containing 15N labeled PCB was ~1 mM. NMR samples were dissolved in buffer containing 10 mM Tris-HCl, pH 7.5, 50 mM KCl, and 7% D2O. Wide-range 1H-15N HSQC, 1H-15N HISQC, and HNN-COSY were measured at 298 K on a Bruker AVANCE III HD 600 spectrometer equipped with a TCI cryogenic probe. In the 1H coupled HISQC experiment, RcaE was dissolved in a buffer of the same composition but not containing D2O, for exclusively observing signals of nondeuterated (e.g., +NH2D, NHD), but protonated isotopomers (e.g., +NH3, NH2). A 4.1 mm o.d. Shigemi tube containing the protein solution was placed into a 5.0 mm o.d. tube containing pure D2O, for the 2H lock signal.

1H-Decoupled 1D 1H-15N INEPT Measurements with or without LOW-BASHD Decoupling.

1 mM 15N-Lys-15N PCB labeled RcaE holoprotein (unlabeled apoprotein incorporating 15N lysine with 15N labeled PCB) was dissolved in buffer containing 10 mM Tris-HCl, pH 7.5, 50 mM KCl, and 7% D2O. NMR spectra with or without homonuclear LOW-BASHD 15N decoupling were recorded on a Bruker Avance III HD 800 MHz spectrometer equipped with a cryogenic TXO probe at 298 K. Simulated 15Nζ signals of Lys261 with a series of various magnitudes of h2JNN were made using the program DNMR (Bruker). In the simulation, the line-width of the signal in the absence of J coupling was set to 5 Hz, which is read from the spectrum of 15Nζ peak recorded with LOW-BASHD decoupling.

1D1H-15N Spin-Echo Difference TROSY.

1 mM 15N-Lys-15N PCB labeled RcaE holoprotein (unlabeled apoprotein incorporating 15N lysine with 15N labeled PCB) was dissolved in buffer containing 10 mM Tris-HCl, pH 7.5, 50 mM KCl, and 7% D2O. 1H-15N Spin-echo difference TROSY spectra were measured at 298 K on a Bruker AVANCE NEO 900 spectrometer equipped with a TCI cryogenic probe.

Isotope Shift Monitoring on 13Cε of Lysine.

The final concentration of RcaE holoprotein incorporating 13Cε labeled lysine and containing unlabeled PCB was ~1 mM. NMR samples were dissolved in buffer containing 10 mM Tris-HCl, pH 7.5, 50 mM KCl, and 0, 50, or 100% D2O. A 4.1 mm o.d. Shigemi tube containing the RcaE solution was inserted into a 5 mm o.d. outer tube containing D2O for the 2H lock signal. 1D 13C experiments were measured at 303 K on a Bruker AVANCE III 600 spectrometer equipped with a DCH cryogenic probe.

QM/MM Calculations.

The atomic coordinates were obtained from the crystal structure of the GAF domain of RcaE in the Pg state (PDB ID: 8K9O). Atomic partial charges of the amino acids were adopted from the all-atom CHARMM22 (33) parameter set. Hydrogen atoms were generated and energetically optimized with CHARMM (34), while heavy atom positions were fixed. During this process, all titratable residues were in their standard protonation states at pH 7 (i.e., acidic groups were ionized, and basic groups were protonated).

The resulting atomic coordinates were used to calculate the protonation pattern of the titratable residues by solving the linear Poisson–Boltzmann equation using the MEAD program (35). In the protein environment, the difference in electrostatic energy between protonated and deprotonated states was calculated and the pKa value experimentally measured in water (e.g., 4.0 for Asp) was added as a reference pKa value (36). The reference pKa values used were 12.0 for Arg, 4.0 for Asp, 9.5 for Cys, 4.4 for Glu, 10.4 for Lys, 9.6 for Tyr (36), and 7.0 and 6.6 for the Nε and Nδ atoms of His, respectively (37–39). All other titratable sites were equilibrated to the protonation state of the target site during titration. Computations were conducted at 300 K, pH 7.0, and with an ionic strength of 100 mM and dielectric constants of four for the protein interior and 80 for water. The linear Poisson–Boltzmann equation was solved using a three-step grid-focusing procedure at resolutions of 2.5, 1.0, and 0.3 Å. The Monte Carlo method with the Karlsberg program (40) was used to sample the ensemble of protonation patterns, yielding probabilities [protonated] and [deprotonated] for the two protonation states of the molecule.

Considering the calculated protonation pattern, hydrogen atoms were generated explicitly for further QM/MM calculations. The geometry was optimized using a QM/MM approach. The restricted density functional theory (DFT) method was employed with the CAM-B3LYP functional (41), 6-31G* basis sets, and the CAM-B3LYP parameter μ of 0.14 (42), using the QSite program version 5.8 (Schrödinger, LLC, NY). All atomic coordinates were fully relaxed in the QM region. In the MM region, hydrogen atom positions were optimized using the OPLS2005 force field (43), with heavy atom positions fixed. Two QM regions were compared: a small QM region with [PCB and the sidechain of Lys261] and a larger QM region with [PCB, the sidechains of Lys261, Cys248, and residues in the H-bond network (i.e., Tyr227, Arg231, Arg233, and His285), and the backbone groups in the H-bond network (Val218, Phe219, Leu245, and Ala246)]. As the results were essentially the same, the results from the small QM region were presented in the main text, unless otherwise specified. See SI Appendix, Table S2 for the results obtained using the large QM region. See Dataset S1 for the atomic coordinates of the QM/MM-optimized geometry.

To obtain the potential-energy profile of the H-bond, the focusing H atom was incrementally moved from the H-bond donor N atom of PCB to the acceptor N atom of Lys261 by 0.05 Å. After each movement, the geometry was optimized by constraining the Ndonor–H and H–Nacceptor distances, and the energy of the resulting geometry was calculated. This process was repeated until the H atom reached the Nacceptor atom. The absorption wavelength of the PCB chromophore was determined using a time-dependent DFT method with the CAM-B3LYP functional (41) [μ =0.14 (42)] and 6-31G* basis set in the GAMESS program (44). A QM/MM approach with the polarizable continuum model (PCM) method was used to calculate the absorption energies, explicitly considering the electrostatic and steric effects of the protein environment in the presence of bulk water (with a dielectric constant of 78 for the bulk region). In the PCM method, polarization points were positioned on spheres with a radius of 2.8 Å from the center of each atom to implicitly represent water molecules in the cavity.

Based on the QM/MM-optimized structures, the NMR chemical shift for 15N (δ) and the NMR spin–spin coupling constant (J) were calculated. The shielding constant σ was calculated at the same level of theory as the geometry optimization by using the pseudospectral GIAO method (45) implemented in the Qsite program (41). The resulting value was converted to δ using the empirical formula (46): δ = 380.23 – (σ + 126.77)/0.9776. J was calculated at the B3LYP/6-31 g level of theory using the GIAO method (47, 48) with the SpinSpin option (49, 50) implemented in the Gausian09 program (51).

Supplementary Material

Appendix 01 (PDF)

Dataset S01 (TXT)

We thank Koshiro Kido for assistance in theoretical calculations, and Dr. Takahisa Ikegami, Satoshi Kamino, Dr. Teppei Kanaba, Dr. Naoya Tochio, and Dr. Toshio Nagashima for helpful discussions about NMR measurements. X-ray crystallography was performed as a project of the Aichi Synchrotron Radiation Center (project 2021N6003), and the NMR experiments were performed in part using NMR spectrometers with ultrahigh magnetic fields under the Collaborative Research Program of Institute for Protein Research, Osaka University, NMRCR-18-05, -19-05, and -20-05, and performed in part at RIKEN NMR Platform supported by the Ministry of Education, Culture, Sports, Science and Technology Program Grant Number JPMXS0450100021. This research was supported by JSPS KAKENHI (JP23H04963 and JP24K01986 to K.S.; JP20H03217 and JP23H02444 to H.I.; JP22H02562 and JP24H02094 to M.M.) and Interdisciplinary Computational Science Program in CCS, University of Tsukuba (K.S.).

Author contributions

M.K. and M.M. designed research; T. Nagae, M.T., T. Noji, K.S., H.A., Y.M., Y.I., Y.H., H.I., and M.M. performed research; H.A. contributed new reagents/analytic tools; T. Nagae, T. Noji, K.S., H.I., and M.M. analyzed data; and H.I. and M.M. wrote the paper.

Competing interests

The authors declare no competing interest.

Data, Materials, and Software Availability

Previously published data were used for this work (figure S3B was created based on data from refs. 15 and 16). All other data are included in the article and/or supporting information.

Supporting Information

This article is a PNAS Direct Submission.
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