NâHÂ·Â·Â·N Hydrogen Bonds Involving Histidine Imidazole Nitrogen

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N-H…N Hydrogen Bonds Involving Histidine Imidazole Nitrogen Atoms: A New Structural Role for Histidine Residues in Proteins Rama Nagesh Venkata Krishna Deepak, and Ramasubbu Sankararamakrishnan Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00253 • Publication Date (Web): 15 Jun 2016 Downloaded from http://pubs.acs.org on June 23, 2016

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N-H…N Hydrogen Bonds Involving Histidine Imidazole Nitrogen Atoms: A New Structural Role for Histidine Residues in Proteins R. N. V. Krishna Deepak and Ramasubbu Sankararamakrishnan* Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur 208016, India

*Author for Correspondence Email: [email protected] Tel: +91 512 259 4014 Fax: +91 512 259 4010

Funding Information: The High Performance Computing (HPC) facility at IIT-Kanpur supported by DST and MHRD is gratefully acknowledged. Krishna Deepak thanks Council of Scientific and Industrial Research (CSIR) for a Senior Research Fellowship.

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Abbreviations PDB: Protein Data Bank; HB: Hydrogen bond; NMA: N-methylacetamide; Ace-GlyNMe: N-acetyl-glycine-N-methylamide; BSSE: Basis set superposition error


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Abstract The amino acid histidine can play a significant role in the structure and function of proteins. Its various functions include enzyme catalysis, metal-binding activity and involvement in cation-π, π-π, salt-bridge and other types of non-covalent interactions. Although histidine’s imidazole nitrogens (Nδ/Nε) are known to participate in hydrogen bond (HB) interactions as acceptor or donor, a systematic study of N-H…N HBs with Nδ/Nε atom as acceptor has not been carried out. In this study, we have examined two datasets of ultra high-resolution (Dataset-I) and very high-resolution (Dataset-II) protein structures and identified 28 and 4017 examples of HBs of N-H…Nδ/Nε type from both datasets involving histidine imidazole nitrogen as acceptor. In nearly 70% of them, main-chain N-H is the HB donor and the majority examples are from the NH group separated by two residues (Ni+2-Hi+2) from histidine. Quantum chemical calculations using model compounds were performed with imidazole and N-methylacetamide (NMA) and they assumed conformations from 19 examples from Dataset-I with N-H…Nδ/Nε HBs. BSSEcorrected interaction energies varied from -5.0 to -6.78 kcal/mol. We also found that imidazole nitrogen of 9% of histidine residues forming N-H…Nδ/Nε interactions in Dataset-II participate in bifurcated HBs. Natural bond orbital analyses on model compounds indicate that the strength of each HB is mutually influenced by the other. Histidine residues involved in Ni+2Hi+2…Nδi/Nεi HBs are frequently observed in a specific N-terminal capping position giving rise to a novel helix-capping motif. Along with their predominant occurrence in loop segments, we propose a new structural role for histidines in protein structures.


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Among the 20 naturally occurring amino acids, histidine (His) is unique and its sidechain imidazole ring can exhibit both aromatic as well as basic character. Histidine residue has been commonly found to be part of catalytic sites of several enzymes. Analysis of small number of 223 annotated enzymes showed that histidine is among the top seven catalytic residues that plays dominate role in catalysis 1. This residue is involved in general acid/base catalysis, covalent catalysis, proton and electron shuttling and stabilization. Histidine is most frequently found to be a coordinating residue in metal-binding sites of many proteins. It acts as a ligand for metal ions like copper, nickel, iron and zinc 2-10. The imidazole ring of histidine also participates in several non-covalent interactions involving the π-electron cloud of its aromatic ring. Its basic character enables the residue to take part in salt-bridge interactions. The proton conduction and gating of tetrameric M2 protein of influenza virus is regulated by the cation-π interactions between histidine and tryptophan residues within the transmembrane region

11

. Analysis of

protein structures from the Protein Data Bank (PDB) revealed that histidine and tryptophan sidechains prefer to interact in parallel orientation

12

. In another PDB structure analysis, π-π

interaction networks involving histidine and other aromatic residues are found more in number in multidomain and membrane proteins and also in lyases

13

. In plastocyanin from a fern plant, it

has been shown that π-π interactions between the active-site histidine and another aromatic residue helps to prevent the active site from being inactivated

14

. The π electron cloud of

histidine residue is also shown to be involved in lone pair-π interactions in several protein structures 15. Similarly, the imidazole nitrogens are known to participate in N-H…π interactions with the π-electron cloud of other aromatic residues 16-17.


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Histidine is one of the three basic residues that can participate in salt-bridge interaction with acidic residues. The salt-bridges formed by histidine residues have been found to play important biological role in many proteins. For example, the pH-dependence of oxygen affinity in hemoglobin

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and the pH-dependent quaternary structure formation of an eubacterial proton

pump Gloeobacter rhodopsin

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seem to be influenced by the salt-bridge interactions of His

residues. DeGrado and his coworkers have recently characterized the geometry and other characteristics of salt-bridges involving histidine along with arginine and lysine residues in detail 20

. In the neutral form, the nitrogen atoms of the imidazole ring can act as hydrogen bond

(HB) acceptor and/or donor. The subatomic resolution structure of the enzyme cholesterol oxidase revealed the positions of hydrogen atoms and enabled to deduce that the active site histidine residue is involved in hydrogen bond (HB) interaction as a HB-donor 21. Similarly, the active site His of human acetylcholinesterase is involved in short and strong hydrogen bond interaction with a Glu residue and the histidine imidazole NH serves as HB donor

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. The

imidazole nitrogen atoms are also reported to take part in HB interactions as HB acceptors in several enzymes such as porcine pancreatic elastase horseradish peroxidase

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23

, ornithine 4,5-aminomutase

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and

. Using quantum chemical calculations, catalytic histidine has been

shown to form hydrogen bond interactions to stabilize the enolate intermediate in citrate synthase 26-28

. The HB interactions involving imidazole ring is mainly of N-H…O and N…H-O types.

Very few studies have reported the existence of hydrogen bond of N-H…N type in proteins. In the case of histidine, recently Juliette Lecomte and her coworkers identified bifurcated hydrogen bonds in ankyrin repeats involving the imidazole nitrogen Nδ as the HB acceptor atom with Thr backbone nitrogen and side-chain oxygen atoms

29

. Histidine also has been shown to play a


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structural role in the N-cap regions of helical structures as demonstrated in truncated hemoglobins and cytochrome b5 using NMR coupling constant

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. McDonald and Thornton

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carried out a systematic study of hydrogen bonds in proteins formed by all 20 residues including histidine by analyzing 57 protein structures with resolution 2 Ǻ or better. However with the enormous increase in the number of structures available in PDB, it would be interesting to find out how frequently histidine imidazole ring participates in HB interactions as HB acceptor and whether there is any structural and functional significance associated with such interactions. In this paper, we have systematically analyzed two datasets of protein structures, Dataset-I with ultra high-resolution structures and Dataset-II with very high-resolution structures. Structures in Dataset-I were determined by neutron diffraction or X-ray diffraction with resolution 0.9 Ǻ or better. Hydrogen positions in the structures of Dataset-I were determined experimentally. Very high-resolution structures in Dataset-II were X-ray diffraction structures with resolution 1.8 Ǻ or better. Such a categorization is necessary because Dataset-I has fewer number of structures and hence it will be difficult to generalize the conclusions based on small number of structures. Dataset-II with large number of structures relies on algorithms to construct hydrogen atom positions. Conclusions based solely on Dataset-II will have to depend on the accuracy of computational methods to build hydrogen atoms. Hence both datasets are needed to reach reliable conclusions. We found that in less than 30% of all hydrogen bonds formed by imidazole hydrogen bonds, the Nδ/Nε atoms act as HB acceptors and significant number of them are formed by the main chain nitrogen atom of i+2 residue and the ith histidine imidazole nitrogen. Such Nδi/Nεi…Hi+2-Ni+2 hydrogen bonds are predominantly found in loops and to some extent in N-terminal capping positions of helical structures. Further characterization by quantum


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chemical calculations using model compounds indicates that the Nδ/Nε…H-N HB in this motif can be a significant factor in stabilizing the loop structures and N-terminal cap of helices.

Materials and Methods Datasets of protein structures The two datasets of protein structures are the same which we used to find the N-H…N hydrogen bonds involving proline residues in our earlier study 32. Briefly, structures belonging to Dataset-I were determined by neutron diffraction or X-ray diffraction (resolution 0.9 Ǻ or better) or hybrid approach that used both neutron and X-ray diffraction methods. Hydrogen atoms in these structures were determined from the experimental methods and are available in the PDB files. Structures from Dataset-II consisted of all high-resolution structures (resolution ≤ 1.8 Ǻ; Rvalue ≤ 0.20; R-free ≤ 0.25) made available as of March 2015. The program CD-HIT

33-34

was

used to remove the redundancy at the level of 30% sequence identity cut-off and REDUCE

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was used to build hydrogen atoms.

N-H…N hydrogen bonds involving histidine imidazole ring Histidine imidazole nitrogen atoms (Nδ and Nε) can serve as an acceptor or donor depending upon its protonation state. In this study, we first identified all the hydrogen bonds in which Nδ/Nε atom(s) are involved. The eight types of hydrogen bonds considered are Nδ/NεH…O, Nδ/Nε-H…N, Nδ/Nε…H-O and Nδ/Nε…H-N. We used the following criteria to define a hydrogen bond

35-36

. The distances d(D…A) and d(H…A) should be less than or equal to 3.5 Ǻ

and 2.5 Ǻ respectively. Two angles θ(D-H…A) and θ(H…A-AA) were considered and they


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should be at least 90°. The notations D, A, H and AA in the above distance and angle parameters represent donor, acceptor, hydrogen atom and acceptor antecedent respectively.

Quantum chemical calculations on model compounds (a) With single N-H…N hydrogen bonds To find out the strength of Nδ/Nε…H-N hydrogen bonds formed by His(i) and the backbone amino group, we performed quantum chemical calculations using model compounds. A representative example of histidine participating in N-H…Nδ/Nε hydrogen bond is shown in Figure 1A. For quantum chemical calculations, we considered the model systems imidazole and N-methylacetamide (NMA) to represent histidine and the backbone amine group respectively (Figure 1B). Both molecules were first geometry optimized individually using the electronic structure program package ORCA v3.0.2

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with BP86 density functional theory

38-39

and

Ahlrichs' triple-zeta def2-TZVP basis set 40-41. The optimized molecules were then independently superposed on the side-chain of histidine and the backbone of the residue that are involved in Nδ/Nε…H-N hydrogen bond from each protein structure in which the HB is found. The interaction energy between the two moieties was calculated using equation (1). Eint(X-Y) = EXY – EX – EY

(1)

Where X and Y represent the imidazole and NMA respectively; EXY is the single point energy of imidazole and NMA groups with Nδ/Nε…H-N hydrogen bond as observed in a given protein structure, EX and EY are single point energies calculated separately for imidazole and NMA respectively. Single point energies were calculated using Gaussian 09 chemistry comprising of M06-2X density functional theory

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42

along with model

in conjunction with Dunning's

correlation-consistent, quadruple zeta basis set augmented with diffuse functions AUG-cc-pVQZ 8 ACS Paragon Plus Environment

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44

. Basis set superposition error (BSSE) was accounted for by employing Boys and Bernardi's

standard counterpoise (CP) correction method 45.

Figure 1: (A) A representative example of a histidine (H306) imidazole ring forming NH…Nδ/Nε hydrogen bond with the main-chain N-H group of i+2 residue (D308) from human aldose reductase (PDB ID: 2R24). The distance between the Nδ of His306 and main-chain N-H hydrogen of D308 is indicated. The molecular modeling software UCSF Chimera 46 is used to draw all molecular plots. (B) The model compounds imidazole and N-methylacetamide used in quantum chemical calculations are shown with the distance between the hydrogen and the imidazole nitrogen participating in N-H…N hydrogen bonds. The coordinates of these model compounds correspond to those of His306 and D308 of 2R24 structure. The distance depicted is after optimizing both the systems individually and superposing them on the PDB conformation.

(b) With bifurcated N-H…Nδ/Nε hydrogen bonds With large number of histidine residues participating in bifurcated hydrogen bonds with at least one of them being N-H…Nδ/Nε, we wanted to find out if the strength of bifurcated hydrogen bonds relative to the same system with single hydrogen N-H…N bond. We performed quantum chemical calculations by considering imidazole representing histidine. We used Nacetyl-glycine-N-methylamide (Ace-Gly-NMe) as a compound that forms two N-H…Nδ/Nε hydrogen bonds with the imidazole moiety (see below). The model systems assumed the conformations observed in the examples found in the protein structures. Before calculating the interaction energy, the hydrogen atoms were first optimized using the model chemistry BP86 3839

density functional theory and Ahlrichs' triple-zeta def2-TZVP 9 ACS Paragon Plus Environment

40-41

basis set. The electronic

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structure program ORCA v3.0.2

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was employed to optimize the hydrogen atoms. BSSE-

corrected interaction energy between the two molecules was calculated in the same way using equation (1) as that used previously. Single point energies EXY, EX and EY were evaluated using the same theory and basis set as described in the previous section.

(c) Natural Bond Orbital (NBO) analyses NBO analysis has been performed on systems containing histidine participating in other interactions 47. In this study, NBO calculations were performed on model compounds imidazole and Ace-Gly-NMe with bifurcated N-H…Nδ/Nε hydrogen bonds and for comparison purpose, control systems with only one N-H…Nδ/Nε hydrogen bond were also used for NBO analyses. The conformations of the systems are the same as described in the previous section. M06-2X density functional theory along with the AUG-cc-pVQZ

44

43

basis set were employed in the

calculations. Stabilization energies were estimated using second order perturbation theory as implemented in NBO version 3.1 48 available in Gaussian 09 42.

Results Identification of N-H…Nδ/Nε hydrogen bonds Two datasets of protein structures, Dataset-I and Dataset-II, were considered to identify HBs formed by imidazole ring of histidine residues. Only those HBs in which the donor and acceptor atoms are nitrogen and/or oxygen are found out. The non-redundant Dataset-I and Dataset-II contained 64 (68 polypeptide chains) and 5336 (5442 polypeptide chains) protein structures respectively. After applying the HB criteria as described in the Methods section, we identified 98 and 20,308 HBs which involved Nδ or Nε atom of imidazole group of histidine.


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Since our interest is to find out the frequency and characterization of N-H…N type of HBs formed by imidazole ring, we narrowed the focus on those HBs in which Nδ/Nε participates in HB interaction with another nitrogen atom as an acceptor or donor. In Dataset-I and Dataset-II, 28 out of 98 (28.5%) and 4017 out of 20,308 (20%) HBs belong to N-H…N type category. Among all N-H..N HBs, 68 to 69% of them (19 out of 28 in Dataset-I and 2830 out of 4017 in Dataset-II) are formed by a main-chain N-H group and histidine Nδ/Nε atom and imidazole nitrogen acts as an acceptor atom. All 19 examples from Dataset-I are summarized in Table 1. It can be seen from this table that main-chain N-H group in 11 out of 19 examples comes from residues separated by two positions from histidine. An example of one such interaction from the protein human aldose reductase (PDB ID: 2R24) is shown in Figure 1A. We also looked at the spatial separation of main-chain N-H acceptor along the primary sequence in Dataset-II with respect to the histidine residue (Figure 2). As in Dataset-I, it is very clear that large number of examples are found in which main-chain N-H group from i+2 position participate in N-H…N HB interaction as HB donor with the imidazole group of ith histidine residue. The geometrical parameters of all N-H…Nδ/Nε HBs are summarized for both datasets in Table 2.


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Figure 2: Spatial separation of residues that provide the main-chain N-H group to participate in N-H…N type of hydrogen bonds with histidine (designated as ith residue) along the primary sequence. Histidine imidazole nitrogen atoms act as HB acceptors.

Quantum chemical calculations on model compounds To characterize and find out the strength of N-H…Nδ/Nε HB interaction between imidazole nitrogen and main-chain N-H respectively as HB acceptor and donor, we performed quantum chemical calculations on model compounds as described in Materials and Methods. Imidazole and N-methylacetamide were used to represent the histidine side-chain and the mainchain N-H respectively (Figure 1B). The coordinates of imidazole and NMA were taken from the examples of 19 ultra high-resolution protein structures from Dataset-I and they were first geometrically optimized. Interaction energy between the two moieties was calculated for each of the 19 cases and they are listed in Table 1. It is clear that the interaction energies between the two compounds do not vary much and the values lie between -5.00 to -6.78 kcal/mol in 16 out of 19 examples. Only in three cases, the energy was positive (+2.5 to +11.0 kcal/mol) due to the fact that both nitrogen atoms of imidazole ring are protonated in the crystal structures (Supplementary Figure S1). This brings both the protonated imidazole nitrogen and the main12 ACS Paragon Plus Environment

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chain N-H group close together and such close contact resulted in unfavorable positive interaction energy. The highly favorable nature of interactions in 16 out of 19 cases indicate that the N-H…Nδ/Nε hydrogen bond indeed can contribute to the stability of protein structures.

Bifurcated hydrogen bonds involving imidazole Nδ/Nε atom as acceptor Using NMR spectroscopy, Lecomte and her coworkers have reported the existence of a bifurcated HBs in which the Nδ atom of histidine imidazole is involved in making HBs simultaneously with the main-chain NH group and the side-chain hydroxyl group of a threonine residue in ankyrin repeats

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. We also looked at the possibility of at least some of the histidine

residues participating in N-H…Nδ/Nε type of interaction to form two HBs at the same time. We found that 3848 histidine residues are involved in 4017 N-H…Nδ/Nε HBs. Among these histidines, we identified 348 residues participating in a second hydrogen bond. A close examination of the bifurcated HBs revealed that in addition to N-H…Nδ/Nε, 169 form an additional N-H…Nδ/Nε hydrogen bond. The second HB in the remaining 179 can be classified as O-H…Nδ/Nε type. A representative example of a protein with bifurcated HBs of N-H…N type with histidine imidazole nitrogen as HB donor is shown in Figure 3A. Thus 9% of total number of histidine residues involved in N-H…Nδ/Nε hydrogen bonds also forms another hydrogen bond simultaneously. Among the 169 bifurcated hydrogen bonds in which both HBs are of N-H…Nδi/Nεi type, the donor N-H groups in 93 examples are contributed by i+2 and i+3 residues (Figure 3A).


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Figure 3: (A) A representative example in which the histidine (H108) imidazole nitrogen forms bifurcated hydrogen bonds and both can be classified as N-H…Nδ type with the HB donors provided by the main-chain N-H groups of i+2 (R110) and i+3 (F111) residues from the protein pseudouridine synthase (PDB ID: 1DJ0). (B) An example of Ni+2-Hi+2…Nδi hydrogen bond formed between the histidine (H474) imidazole Nδ and the main-chain N-H of V476 with the proline (P475) present as i+1 residue from the protein endonuclease (PDB ID: 2NRT). (C) A representative example of histidine (H264) at the N-cap position of an α-helix participating in Ni+2-Hi+2…Nδi hydrogen bond with the main-chain N-H group of i+2 residue (T266) occupying the N2 position within the N-terminal capping region of a bacterial vitamin C transporter (PDB ID: 4RP9). (D) An example of a histidine (H495) stabilizing a loop segment by participating in N-H…Nδ hydrogen bond with the main-chain N-H of i+2 residue (I497) from an enzyme farnesyltransferase (PDB ID: 4L9P).

To investigate whether such bifurcated HBs actually enhance the stability, we performed quantum chemical calculations using model compounds. As in the previous case, histidine was represented by the imidazole group and the two N-H groups from successive residues were represented by N-Acetyl-glycine-N-methylamide (Ace-Gly-NMe) (Figure 4). We considered two examples of bifurcated HBs and for comparison purpose we also carried out calculations on the


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same model compounds with only one N-H…Nδ/Nε hydrogen bond. His421 and His154 respectively from alkaline phosphatase (PDB ID: 3E2D) and a putative phosphatase (PDB ID: 3DAO) structures were considered for bifurcated HBs. They both form N-H…Nδ/Nε hydrogen bonds with the main-chain N-H groups of i+2 and i+3 residues simultaneously. The residues His485 and His269 respectively from human acyl-CoA synthetase (PDB ID: 3C5E) and cyclic nucleotide phosphodiesterase (PDB ID: 1ZKL) form only one N-H…Nδ/Nε HB with the mainchain N-H group of i+2 residue. The coordinates for the model compounds were directly imported from the respective PDB structures. Since the proteins were from Dataset-II, the hydrogen atoms were built using REDUCE

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and the program MOLDEN

49

was used to cap

Ace-Gly-NMe molecule with methyl groups. The hydrogen atoms of the two molecules were first optimized as described in the Materials and Methods section. Single point energy calculations were performed on the model systems and BSSE-corrected interaction energies are 11.43 and -12.63 kcal/mol respectively when the coordinates were taken from PDB structures 3E2D and 3DAO. As in the previous calculations with single N-H…N hydrogen bond, these calculations again demonstrate that the N-H…N HBs can be a stabilizing factor. We also performed additional calculations with examples in which only a single N-H…N HB using the same model compounds, namely, imidazole and Ace-Gly-NMe (Figure 4). The two examples were H485 and H269 from human acyl-cosynthetase (PDB ID: 3C5E) and cyclic nucleotide phosphodiesterase (PDB ID: 1ZKL). After the PDB coordinates corresponding to these regions were imported to the model compounds (Figure 4B) and optimizing the hydrogen atoms, the calculated interaction energies are -9.43 and -9.18 kcal/mol respectively. Thus the system with the bifurcated HBs (two N-H…N HBs) is energetically more favorable by 2 to 3.4 kcal/mol compared to the system with just one N-H…N hydrogen bond. This indicates that bifurcated


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HBs of the type N-H…N can provide additional stability to the system. In the case of His485 and His269 respectively from 3C5E and 1ZKL, it is possible that small dynamical motions can bring the N-H group of i+3 residue closer to the histidine imidazole nitrogen so that bifurcated hydrogen bonds can be formed.

Figure 4: (A) and (B) Model compounds with bifurcated hydrogen bonds of N-H…Nδ/Nε type. Coordinates correspond to the residues (A) H421, Y423 and L424 from the enzyme alkaline phosphatase (PDB ID: 3E2D) and (B) H154, D156 and K157 from a putative phosphatase structure (PDB ID: 3DAO). (C) and (D) represent the same model compounds with a single NH…Nδ/Nε hydrogen bond. The coordinates of (C) H485, A487 and V488 from the protein human acyl co-synthetase (PDB ID: 3C5E) and (D) H269, L271 and A272 from a cyclic nucleotide phosphodiesterase (PDB ID: 1ZKL) were considered for quantum chemical calculations. The distances between the imidazole nitrogen and the backbone N-H groups of i+2 and i+3 residues are displayed. The BSSE corrected interaction energies obtained from quantum chemical calculations are shown for each system.


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To further characterize these interactions, we performed Natural Bond Orbital (NBO) analyses on the above systems and the results of NBO calculations are presented in Table 3. The occupancy of Nδ/Nε lone pairs and the non-Lewis orbitals of Ni+2-Hi+2 and Ni+3-Hi+3 are provided along with the Natural Population Charges (NPA) charges.

The second order

perturbation energy due to orbital overlap of n σ* interactions are also mentioned in Table 3. For both examples having bifurcated HBs, the stabilization energy is very significant ranging from 7.1 to 13.6 kcal/mol. For the same model compounds when only the Ni+2-Hi+2…Nδi/Nεi hydrogen bond is present and the Ni+3-Hi+3…Nδi/Nεi HB is absent, NBO analyses was performed. The calculations show that the strength of Ni+2-Hi+2…Nδi/Nεi is 16.6 to 19.4 kcal/mol which is significantly higher than that observed for bifurcated hydrogen bonds. This is also reflected in the lower occupancy of anti-bonding orbitals corresponding to Ni+2-Hi+2 bond in 3C5E and 1ZKL.

Secondary structural preferences for N-H…N HB-forming histidine residues In Dataset-II, almost 50% of all N-H…Nδ/Nε HBs are formed between histidine imidazole nitrogen and main-chain N-H group of i+2 residues. In Dataset-I also 11 out of 19 NH…Nδ/Nε hydrogen bonds are formed between histidine and i+2 residues. Hence, we wanted to find out if there is any specific secondary structural preference for histidine residues involved in Nδi/Nεi…Ni+2-Hi+2 hydrogen bonds with i+2 residues. DSSP method

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as available in Chimera

software 46 was used to assign secondary structures for all the protein structures of Dataset-I and Dataset-II. This is to ensure that the definition used for the secondary structures is uniform across all the structures considered in this study. Table 4 summarizes the frequency of histidine residues in helix, strand or loop regions of protein structures. When main-chain N-H group forms N17 ACS Paragon Plus Environment

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H…N type of HB with the imidazole nitrogen as acceptor, there is a clear preference for the loop region. If only the histidine residues involved in Nδi/Nεi…Ni+2-Hi+2 HBs are considered, then they are overwhelmingly present only in the loop region and only few examples are found in helical secondary structures. We also looked at whether there is any residue preference for the i+1 position when the imidazole nitrogen atom takes part in HB interaction with the main-chain amino group of i+2 residue. We found that proline is present in more than 40% of the examples and percentage of the rest of 19 residues in i+1 position range from 0.4 to 6.8%. Hence, we grouped the examples with proline in i+1 position separately and found out their secondary structural preferences. Nearly all such examples are found in the loop region. A representative example with Nδi/Nεi…Hi+2-Ni+2 hydrogen bond with proline as i+1 residue is shown in Figure 3B. As a control dataset, we considered all the histidines and found out their secondary structural preferences. Only 43% of all histidines prefer to occur in the loop region while 36% and 20% occur respectively in helical and strand regions. We further extended this analysis to find out if histidine residues forming N-H…Nδ/Nε hydrogen bonds have any specific preference for any of the capping positions of secondary structures. The definition of capping positions is the same as that used in our previous analysis 51 and follows the criteria used by Aurora and Rose

52

. The amino-terminal capping positions

consisted of N’’, N’, N-cap, N1, N2 and N3 where N1 to N3 positions belong to the proper secondary structure and N’’, N’ and N-cap positions fall just outside the secondary structure. Similarly, the carboxy terminal capping positions C’’, C’, C-cap, C1, C2 and C3 were considered in which C1 to C3 positions fall within the proper secondary structure. More than 20% (285 out of 1388) of all the histidine residues with Ni+2-Hi+2..Nδi/Nεi HBs are found in N-cap position of helical secondary structures. This increases to 25% (147 out of 581) if we consider only those


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histidine residues which take part in Ni+2-Hi+2..Nδi/Nεi type of HBs in which the i+1 residue is proline. Only 2.1% (525 out of 25238) of all histidine residues which do not engage in Ni+2Hi+2…Nδi/Nεi HB (control data) with the i+2 residue occur in N-cap position. The role of histidine in stabilizing α-helix N-caps has been demonstrated recently in ankyrin repeats

29

,

truncated hemoglobins and cytochrome b5 30 using NMR spectroscopy. In an α-helix, the amino group of the first three residues do not participate in intra-helical HBs and they are available for interactions. Thus when a histidine residue occurs in N-cap position, its imidazole ring can form N-H…N HB with the backbone NH group of residue at N2 capping position (separated by two residues from N-cap position) in a helix. Such an interaction can provide stability to the Nterminal region of helical structures. An example of a histidine residue at N-cap position taking part in Ni+2-Hi+2..Nδi/Nεi HB interaction with the residue at N2 position is shown in Figure 3C. Proline is known to have higher preference to occur in the beginning of helices and if proline occurs in N1 position, then the probability of histidine with Ni+2-Hi+2..Nδi/Nεi HB occurring at N-cap position is even higher. We identified 581 out of 1388 examples of Ni+2-Hi+2..Nδi/Nεi hydrogen bond in which proline is present as i+1 residue. This is about 41% of all histidines forming Ni+2-Hi+2..Nδi/Nεi HBs. Interestingly, when all histidine residues not involved in NH…Nδ/Nε HBs were considered, more than 35% were found in the middle of helical structures. However, histidines participating in Ni+2-Hi+2..Nδi/Nεi completely avoid occurring in the middle of helices. To find out the side-chain conformational preference for histidine residues that are engaged in Ni+2-Hi+2..Nδi/Nεi type of hydrogen bonds, we plotted the side-chain dihedral angles χ1 versus χ2 and compared them with all those histidines which are not involved in N-H…N hydrogen bonds. We see two distinct populations of histidine side-chains involved in Ni+219 ACS Paragon Plus Environment

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Hi+2..Nδi/Nεi hydrogen bonds (Supplementary Figure S2). They are populated around (χ1 = 180°, χ2 = -60°) and (χ1 = +60°, χ2 = +60°). We have superposed the histidines involved in Ni+2-Hi+2..Nδi/Nεi HBs from ultra-high resolution structures of Dataset-I (Figure 5). One can clearly see that both side-chain conformations of histidine can bring its imidazole nitrogen close to the i+2 residue to form Ni+2-Hi+2..Nδi/Nεi hydrogen bonds.

Figure 5: Superposition of histidine residues participating in Ni+2-Hi+2…Nδi/Nεi type of hydrogen bonds plotted from ultra high-resolution structures of Dataset-I. The two populations of histidine side-chains represent the two different clusters of side-chain dihedral angles χ1 and χ2 (see Supplementary Figure S2).

The number of histidine residues with Ni+2-Hi+2..Nδi/Nεi hydrogen bonds in a protein will indicate how much these residues can contribute to the stability of that protein in addition to other potential roles in the function. In our Dataset-II, we found 1093 proteins having at least one histidine participating in Ni+2-Hi+2..Nδi/Nεi hydrogen bond. Among them 236 proteins have two or more histidine residues engaged in the same type of interactions. We have plotted an example of a protein with maximum number of 7 such histidine residues in Figure 6.


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Figure 6: The protein (PDB ID: 3WMU) with maximum number of histidine residues participating in Ni+2-Hi+2…Nδi/Nεi hydrogen bonds.

Discussion Several interactions involving histidine residues including cation-π, π-π, hydrogen-π and metal coordinating interactions have been recently investigated using quantum chemical calculations 53. In this study, it has been shown that histidine’s interaction with metal ions is the strongest followed by cation-π and π-π interactions. Histidine is also found to be more frequently involved in lone pair…π interactions among all the aromatic residues in proteins 15. Scheiner and his group calculated the strengths of different type of HBs formed by the four naturally occurring aromatic residues and compared them

54

. The hydrogen bonds O-H…O, O-H…N, C-H…O and

O-H…π were considered in this study for comparison. Previous studies have searched small molecule structures and biomolecules and analyzed HBs with nitrogen as acceptor including


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those between two amino groups

55-56

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. However to our knowledge, there is no systematic study

of N-H…N hydrogen bonds in which the imidazole nitrogen atoms of histidine side-chain participate as hydrogen bond acceptor. Unlike nucleic acid structures, the N-H…N hydrogen bonds are in general not frequently observed in proteins. In a recent analysis of protein structures 32

, six different hydrogen bonds (N-H…O, O-H…O, N-H…N, O-H…N, C-H…O and C-H…N)

were analyzed and only in about 2% hydrogen bonds, nitrogen acts as acceptor. The fraction of N-H…N hydrogen bonds among all the six hydrogen bonds analyzed is only 1.1%. The imidazole group in histidine can participate in N-H…N hydrogen bonds as acceptor or donor depending upon its protonation state. The systematic analysis of N-H…N hydrogen bonds formed by the imidazole moiety of histidine residues in protein structures has provided many interesting insights regarding its frequency, strength and the secondary structural preferences. Among all hydrogen bonds formed by the imidazole nitrogen atoms, 20 to 28% are formed with imidazole nitrogen as acceptor atom. In majority of these hydrogen bonds, the donor N-H group comes from main-chain. All the 19 N-H…Nδ/Nε hydrogen bond examples of ultra highresolution structures from Dataset-I were subjected to quantum chemical calculations and the model compounds imidazole and NMA were used for this purpose. We found that 16 out of 19 show energetically favorable interaction energy in the range of -5.0 to -6.78 kcal/mol. In the remaining three, imidazole exists in the protonated form and as a result, the interaction energy is positive. When all histidine residues with the respective imidazole nitrogen atoms participating in N-H…Nδ/Nε type of HBs were examined, we found that 9% of them are also simultaneously involved in another hydrogen bond. In almost half of them, the second HB is also N-H…Nδ/Nε type. We further characterized them by performing quantum chemical calculations and NBO


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analyses. The model compound imidazole and Ace-Gly-NMe were used for this purpose. Two examples with bifurcated HBs of N-H…Nδ/Nε type were considered. For comparison purpose, the same calculations were also carried out with the same systems and with only one NH…Nδ/Nε hydrogen bond. Single point energy calculations revealed that the system with bifurcated HBs are 2 to 3 kcal/mol more stable than that with single N-H…Nδ/Nε hydrogen bond. The NBO analyses showed that the second order perturbation energy between the HBparticipating groups is between 7.1 to 13.6 kcal/mol in the case of bifurcated HBs whereas the same for the overlapping orbitals that form a single N-H…Nδ/Nε HB is between 17 to 19 kcal/mol. This indicates that the strength of each hydrogen bond in the case of bifurcated HBs is perhaps reduced due to the influence of the other. Several studies have used quantum chemical calculations on model compounds to characterize and to compare the stability of various types of HBs

57-58

. These studies have also

investigated HBs in which nitrogen is acceptor. The strength of such HBs varied from -5.77 to 36.26 kcal/mol

58

. Scheiner and his coworkers have specifically focused on different types of

HBs formed by aromatic residues

54

. They used water and model compounds representing the

four different aromatic residues. The types of HBs they have studied include O-H…O, N-H…O, C-H…O and O-H…π hydrogen bonds. The strengths of these HBs formed by the aromatic residues were compared. The weakest C-H…O hydrogen bond (1.1 kcal/mol) was reported between benzene and water molecule. The N-H…O hydrogen bond between protonated imidazole and water is the strongest with 15.9 kcal/mol. Similarly in a recent publication, histidine’s multiple roles involving its aromatic π-electron cloud (cation-π and π-π), its interaction with other aromatic residues (hydrogen-π and cation-π) and coordinate interactions with metal ions were investigated using quantum chemical calculations 23 ACS Paragon Plus Environment

53

. However, the HB

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formed by imidazole nitrogen as acceptor was not investigated in these studies. By systematically analyzing the ultra high-resolution and very high-resolution protein structures, the present study not only recognized that a significant number of imidazole nitrogen atoms of histidine residues participate in N-H…Nδ/Nε type of hydrogen bonds, but emphasized the importance of such HBs formed between the N-H group of main-chain and the Nδ/Nε atom of imidazole. The interaction energy (-5.0 to -6.5 kcal/mol) calculated between the model compounds, imidazole and NMA, is comparable to other studies reported in the literature

57-58

and these hydrogen bonds seem to impart stability to the protein structures (see below). We have also found that 9% of histidine residues involved in N-H…Nδ/Nε HBs are also participating in a second HB simultaneously. This second HB can be either O-H…Nδ/Nε or NH…Nδ/Nε type. Quantum chemical calculations on model compounds, imidazole and Ace-GlyNMe, illustrated that the bifurcated HBs provide additional stability in the range of 2 to 3 kcal/mol. NBO analyses clearly showed that strength of the second order perturbation energies of individual HBs in the systems with bifurcated HBs is influenced by each other. Histidine residues involved in N-H…Nδ/Nε hydrogen bond interactions have shown specific preference to occur in the loop regions and in the N-cap position of α-helices. This preference is pronounced if the donor N-H group is contributed by residues which are two positions apart from histidine (i+2) with a proline residue occupying the middle position (i+1). Several N-terminal capping motifs have been identified and their possible role in biological function has been investigated and Bansal

63

52, 59-62

. In a recent study of membrane protein structures, Shelar

have reported an example of a histidine residue participating in helix-capping

interaction in a transmembrane helix of a membrane protein. The current study has clearly shown the frequent instances of histidine residues at the N-cap position participating in N-H…N type of 24 ACS Paragon Plus Environment

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hydrogen bond with main-chain N-H group as HB donor at N2 position in helices. Our systematic analysis has identified this novel N-terminal capping motif involving histidine residues. The energy contribution of the N-H…Nδ/Nε hydrogen bond interactions certainly suggest that these histidine residues can provide stability to the helical segments and loop regions. By mutating the histidine in the N-cap position of α-helices or loop segments, we should be able to deduce the stabilizing role of histidine residues participating in the N-H…Nδ/Nε hydrogen bonds. This study has unraveled a new structural role for histidine along with multitude of other functions carried out by this important residue in proteins.

Supporting Information Supporting Information is available free of charge on the ACS Publications website http://pubs.acs.org. Examples of histidine residues with N-H…Nδ/Nε hydrogen bonds from ultra high-resolution structures with positive interaction energy and χ1 versus χ2 plots of histidine residues with Ni+2Hi+2…Nδi/Nεi hydrogen bonds and without N-H…N hydrogen bonds

Acknowledgements We thank all our lab members for discussion.


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(55) Allen, F. H.; Bird, C. M.; Rowland, R. S. (1995) Correlation of the hydrogen-bond acceptor properties of nitrogen with the geometry of the Nsp2 --> Nsp3 transition in R1(X=)C-NR2R3 substructures: Reaction pathway for the protonation of nitrogen. Acta Cryst. B 51, 1068-1081. (56) Luisi, B.; Orozco, M.; Sponer, J.; Luque, F. J.; Shakked, Z. (1998) On the potential role of the amino nitrogen atom as a hydrogen bond acceptor in macromolecules. J. Mol. Biol. 279, 1123-1136. (57) Grabowski, S. J. (2001) Ab initio calculations on conventional and unconventional hydrogen bonds Study of the hydrogen bond strength. J. Phys. Chem. A 105, 10739-10746. (58) Raub, S.; Marian, C. M. (2007) Quantum chemical investigation of hydrogen-bond strengths and partition into donor and acceptor contributions. J. Comput. Chem. 28, 1503-1515. (59) Chakrabartty, A.; Doig, A. J.; Baldwin, R. L. (1993) Helix capping propensities in peptides parallel those in proteins. Proc. Natl. Acad. Sci. U.S.A. 90, 11332-11336. (60) Lanigan, M. D.; Tudor, J. E.; Pennington, M. W.; Norton, R. S. (2001) A helical capping motif in ShK toxin and its role in helix stabilization. Biopolymers 58, 422-436. (61) Sweeting, B.; Brown, E.; Khan, M. Q.; Chakrabartty, A.; Pai, E. F. (2013) N-terminal helix-cap in αhelix 2 modulates β-state misfolding in rabbit and hamster prion proteins. PLoS One 8, Art. No.: e63047. (62) Lama, D.; Sankararamakrishnan, R. (2011) Molecular dynamics simulations of pro-apoptotic BH3 peptide helices in aqueous medium: Relationship between helix stability and their binding affinities to the anti-apoptotic protein Bcl-XL. J. Compu. -Aided Mol. Des. 25, 413-426. (63) Shelar, A.; Bansal, M. (2014) Sequence and conformational preferences at termini of α-helices in membrane proteins: Role of the helix environment. Proteins: Struct. Func. Bioinf. 82, 3420-3436.


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Table 1: Histidine imidazole nitrogen atoms participating in N-H…N type of hydrogen bonds as acceptor with the main-chain N-H group in ultra high-resolution structures from Dataset-I S. No.

PDB ID/ Chain IDa

Histidine residueb

Donor residuec

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

1GCI/A 1VYR/A 1VYR/A 2GVE/A 2R24/A 2R24/A 3IP0/A 3VXF/H 3W5H/A 3ZOJ/A 4U9H/L 1GCI/A 4F1V/A 2B97/A 2WUR/A 1VYR/A 2WUR/A 2WUR/A 4U9H/L

H39 H103 H181 H96 H187 H306 H115 H91 H1089 H194 H351 H120 H1057 H42 H217 H189 H148 H81 H338

D41 G105 A183 V98 Y189 D308 D117 R93 K1091 A196 W353 K27 N1003 T30 D210 G185 R168 D197 D511

a

d(H…Nδ/Nε) θ(N-H…Nδ/Nε) (Ǻ)d (°)e 2.06 2.14 2.48 2.07 2.45 2.10 2.28 2.24 2.13 2.24 2.26 2.04 2.05 2.20 2.00 2.09 2.13 2.20 2.30

163.4 155.8 153.7 171.1 142.8 171.7 159.9 160.9 160.1 147.5 165.3 174.6 165.9 170.0 156.1 149.9 163.4 165.6 177.1

Interaction energy (kcal/mol)f -6.75 -5.32 -6.03 -6.76 -5.40 -6.78 -5.50 -5.99 -6.11 -5.59 +11.05 -6.60 -6.50 -6.44 -5.00 -5.50 -6.67 +6.47 +2.51

The four letter unique PDB ID and the chain ID are given

b

Residue number of histidine whose imidazole nitrogen participates in N-H…N hydrogen bond as acceptor atom

c

Residue whose main-chain N-H group participates in N-H…N hydrogen bond as donor

d

The distance between the hydrogen and imidazole nitrogen atom Nδ/Nε involved in N-H…N hydrogen bond

e

The angle between donor nitrogen, hydrogen and acceptor nitrogen

f

Interaction energy calculated between the model compounds as described in the Materials and Methods section. The first 11 examples belong to the category of Ni+2-Hi+2…Nδi/Nεi hydrogen bonds. In the remaining 8 cases, the HB donor residues are separated from the acceptor His by more than 2 residues.


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Table 2: Geometrical parameters of N-H…Nδ/Nε hydrogen bonds involving mainchain N-H groups and imidazole nitrogen atoms of histidine residues Parameter d(N…Nδ/Nε) (in Ǻ)b d(H…Nδ/Nε) (in Ǻ)b θ(N-H…Nδ/Nε) (in °)c θ(H…Nδ/Nε-AA) (in °)c, d

Dataset-I (19)a 3.01 ± 0.12 2.18 ± 0.13 161.8 ± 9.3 130.3 ± 10.7

a

The number of examples found in both datasets are given in brackets

b

d(A…B) indicates the distance in Ǻ between atoms A and B

c

Dataset-II (2830)a 3.01 ± 0.11 2.19 ± 0.12 161.9 ± 10.4 127.1 ± 10.9

θ(A-B…C) and θ(A…B-C) indicates the angle in degrees between the atoms A, B and C

d

AA is acceptor antecedent. When Nδ is the HB acceptor atom, AA can be either Cγ or Cε of imidazole ring depending upon which angle satisfies the hydrogen bonding criteria as defined in the Materials and Methods section. Similarly for Nε, AA can be Cδ or Cε of imidazole ring.


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Table 3: Natural Bond Orbital (NBO) analysis of model compounds with bifurcated hydrogen bonds PDB ID 3E2Da 3DAOa 3C5Eb 1ZKLb

NPA chargesc Ni+2 Hi+2 Nδi/Nεi Ni+3 Hi+3 -0.53694 -0.65363 0.43285 -0.64885 0.42683 -0.53746 -0.65773 0.43342 -0.64570 0.43226 -0.51028 -0.67998 0.45027 -0.62029 0.40696 -0.50916 -0.67420 0.44816 -0.62876 0.40620

Orbital occupancy Int. nσ*i+2 nσ*i+3 lp(Ni) σ*(Ni+2-Hi+2) Energyd σ*(Ni+3-Hi+3) (kcal/mol) (kcal/mol)e 1.89697 0.03442 -11.43 9.77 0.03651 8.72 1.89284 0.03123 -12.63 13.62 0.04479 7.11 1.89587 0.01800 -9.43 19.39 0.04992 < 0.05 1.89975 0.01764 -9.18 16.58 0.04665 < 0.05

a

The imidazole nitrogen atoms of H421 and H154 from the protein structures with PDB IDs 3E2D and 3DAO respectively participate in bifurcated hydrogen bonds of N-H…Nδ/Nε type with the main-chain N-H groups from i+2 and i+3 residues. For further details, see Figure 4 and the text. The model compounds imidazole and N-acetyl-glycine-N-methylamide (Ace-Gly-NMe) assumed conformations of these residues from the respective protein structures in quantum chemical calculations. b

The imidazole nitrogen atoms of H485 and H269 from the protein structures with PDB IDs 3C5E and 1ZKL respectively participate in a single N-H…Nδ/Nε type of hydrogen bond with the mainchain N-H of i+2 residue. These examples were used as control in the study of bifurcated hydrogen bonds. For further details, see Figure 4 and the text. The conformations of these residues from the respective protein structures were considered for the model compounds imidazole and Ace-Gly-NMe in the quantum chemical calculations. c

NPA (Natural Population Analysis) charges obtained from NBO analyses are reported for the atoms that are involved in N-H…Nδ/Nε type of hydrogen bonds.

d

BSSE corrected interaction energies calculated between the model compounds.

e

nσ*i+2 and nσ*i+3 are the second order perturbation energies corresponding to the hydrogen bonds of the type Ni+2-Hi+2…Nδi/Nεi and Ni+3-Hi+3…Nδi/Nεi.


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Biochemistry

Table 4: Secondary structure preferences of histidine residues participating in N-H…Nδ/Nε type of hydrogen bonds from Dataset-II Type of hydrogen bond N-H…Nδ/Nε (2830)a Ni+2-Hi+2…Nδi/Nεi (1388)b Ni+2-Hi+2-Proi+1-Nδi/Nεi (581)c All Histidines (25238)

Helix (%)d 18.4 0.8 0.0 36.4

Strand (%)d 11.6 9.9 2.2 20.4

Loop (%)d 70.0 89.3 97.8 43.2

a

All histidine residues involved in N-H…Nδ/Nε type of hydrogen bonds with imidazaole nitrogen (Nδ/Nε) as acceptor and the main-chain N-H group as hydrogen bond donor.

b

Histidine residues participating in N-H…Nδ/Nε hydrogen bonds with the main-chain N-H group separated by two residues

c

Histidine residues that take part in N-H…Nδ/Nε hydrogen bonds with the main-chain N-H group separated by two residues and a proline residue present in between. d

The secondary structures of all protein structures were uniformly assigned using the DSSP algorithm 50 as implemented in the Chimera software 46.


Biochemistry

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For Table of Contents Use Only Manuscript Title: N-H…N Hydrogen Bonds Involving Histidine Imidazole Nitrogen Atoms: A New Structural Role for Histidine Residues in Proteins Authors: R. N. V. Krishna Deepak and Ramasubbu Sankararamakrishnan*


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NâHÂ·Â·Â·N Hydrogen Bonds Involving Histidine Imidazole Nitrogen

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