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Protein Science (2007), 16:1783-1787. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society
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PROTEIN STRUCTURE REPORT

Structure in an extreme environment: NMR at high salt

Bulent Binbuga, Arezue F.B. Boroujerdi, and John K. Young

Department of Chemistry, Mississippi State University, Mississippi State, Mississippi 39762, USA

(RECEIVED April 18, 2007; FINAL REVISION May 21, 2007; ACCEPTED May 23, 2007)


   Abstract
TOP
 Abstract
Introduction
 Results and Discussion
 Conclusion
 Materials and Methods
 Acknowledgments
 References
 
Proteins from halophiles have adapted to challenging environmental conditions and require salt for their structure and function. How halophilic proteins adapted to a hypersaline environment is still an intriguing question. It is important to mimic the physiological conditions of the archae extreme halophiles when characterizing their enzymes, including structural characterization. The NMR derived structure of Haloferax volcanii dihydrofolate reductase in 3.5 M NaCl is presented, and represents the first high salt structure calculated using NMR data. Structure calculations show that this protein has a solution structure which is similar to the previously determined crystal structure with a difference at the N terminus of beta3 and the type of beta-turn connection beta7 and beta8.

Keywords: Haloferax volcanii; dihydrofolate reductase; high salt; NMR; structure calculation


   Introduction
TOP
 Abstract
 Introduction
Results and Discussion
 Conclusion
 Materials and Methods
 Acknowledgments
 References
 
Archaea can survive in extreme conditions such as high-pressure, high-temperature, and hypersaline environments (Jaenicke 1991). Halophilic archaea can thrive in extreme salt medium. The concept of halophilic adaptation has been investigated through studies of the enzyme dihydrofolate reductase (DHFR) (Bohm and Jaenicke 1994b; Bohm 1996). DHFR catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF) by employing the coenzyme nicotinamide adenine dinucleotide phosphate, NADPH (Bohm 1996). The activity and stability of Haloferax volcanii dihydrofolate reductase, hvDHFR1 (162 aa, 17.9 kDa), and mesophilic Escherichia coli dihyrofolate reductase, ecDHFR (159 aa, 17.9 kDa) have been studied over a wide range of salt concentrations (Wright et al. 2002). While there is a linear increase in activity of hvDHFR1 as salt is increased, ecDHFR showed an exponential decrease in activity as salt is increased. Lowering salt concentration on hvDHFR1 from 3.5 M to 0.6 M has an effect on the protein stability, but not on its conformation. However, increasing salt seems to increase the stability of ecDHFR, but again, there is no effect on the structure (Wright et al. 2002).

The X-ray crystallography derived structure of hvDHFR1 (Pieper et al. 1998) (in 2.4 M phosphate buffer) has a Rossman fold similar to that of ecDHFR (Bystroff and Kraut 1991). This represents the only structural information available for hvDHFR1. In contrast, ecDHFR has been studied extensively by X-ray crystallography and NMR in binary as well as ternary complexes at low salt, but not at high salt (Bystroff and Kraut 1991; Falzone et al. 1994; Reyes et al. 1995; Lee et al. 1996; Sawaya and Kraut 1997). These studies revealed that ecDHFR has a flexible catalytic loop (A9–L24) centered around M20 (equivalent to L21 in hvDHFR1) which adopts specific conformations in different complexes (McElheny et al. 2005). NMR relaxation studies have shown that this loop changes mobility upon substrate binding (Osborne et al. 2001; Boehr et al. 2006). It has been suggested that changes in the mobility of the loops may also be connected to changes in salt concentrations (Mevarech et al. 2000; Wright et al. 2002).

Very few proteins from halophilic organisms have been investigated by X-ray crystallography or NMR. Only Halobacterium salinarum Ferredoxin (HsFdx) has been studied by NMR at 0.45 M salt (Marg et al. 2005). There have been no complete relaxation studies at high-salt or at varied-salt concentrations. Appropriate molar salt for halophilic proteins is a vital requirement for these proteins to fold in their native forms, and also for structural stability and activity. Haloferax volcanii flourishes in an extremely high-salt environment, the Dead Sea, which has 3–4 M salt concentration (Pundak et al. 1981). There is a need for more structural data of halophilic proteins for a better understanding of the conformational features that relate to haloadaptation. Here, we present the three-dimensional liquid state NMR-derived structure of hvDHFR1 at 3.5 M NaCl. This represents the first protein studied by NMR at such an extreme salt concentration.


   Results and Discussion
TOP
 Abstract
 Introduction
 Results and Discussion
Conclusion
 Materials and Methods
 Acknowledgments
 References
 
NMR and high salt
Cosolute concentration can have an effect on the conformation of a protein. In order to determine if the conformation of hvDHFR1 changes at different salt concentrations a series of 15N-HSQC were acquired from 1.0 M to 3.5 M NaCl in 0.5 M increments (data not shown). As the salt is increased, there is a uniform shift of all peaks in the 15N-HSQC to a larger 1H and 15N chemical shift. These data suggest that there is no major shift in the overall conformation of hvDHFR1 with increasing salt, which is also supported by the previously published circular dichroism and fluorescence data (Wright et al. 2002).

Protein structure determination
A total of 185 {Phi} and {psi} dihedral angles were derived from TALOS and used in the structure calculations and restricted within the intervals of ±20°. The assigned 1371 NOE 1H-1H distance restraints from the NOESY experiments were employed in the CNS structure calculation (Table 1). Four NOE peak intensity categories were used to classify intra 1H-1H upper limit distances: strong (<2.7 Å), medium (<3.3 Å), weak (<4.0 Å), and very weak (<4.5 Å). The presence of a cis peptide bond between two glycine residues has been identified in the other DHFR enzymes by X-ray crystallography (Reyes et al. 1995). The existence of this cis peptide bond for hvDHFR1 (G101–G102) was supported by the strong 1H{alpha} G101 to 1H{alpha} G102 and strong 1HN G45 to 1HN G102 NOE correlations. This cis peptide geometry was maintained by defining a restraint of 0° for the dihedral angle C{alpha} G101 to C{alpha} G102, with an energy constant of 5 kcal mol–1 rad–2. This constraint caused an improper violation in the structures output from the CNS. Therefore, the structures were further minimized using the Discover module of the InsightII program (version 2005, Accelrys). Hydrogen bonds were added (based on previous calculations) in secondary structure regions for the last few rounds of calculations. Lyophilized hvDHFR1 cannot be redissolved; therefore, H/D exchange experiments were not obtained. Once all violations were eliminated, 100 structures were generated for final analysis. The 20 lowest energy structures for hvDHFR1 are shown in Figure 1A and the statistics given in Table 1. The final family of structures exhibits a good convergence with a root mean square deviation (RMSD) value of 0.83 ± 0.27 Å for backbone and 0.37 ± 0.12 Å for secondary structure regions, respectively. The quality of the structures was evaluated using PROCHECK, which showed that 96.2% of the residues fall in allowed and additionally allowed regions of the Ramachandran plot (Laskowski et al. 1993). A representative NMR-derived structure, Figure 1B, reveals that four helices are packed across a central beta-sheet with eight beta-strains. The atomic coordinates have been deposited in the RCSB protein data bank under the code of 2ITH.


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  		Table 1. Experimental constrains and statistics of the 20 lowest energy structures calculated by CNS

 


Figure 1
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  		Figure 1. (A) Backbone superimposition of the 20 lowest energy structures of hvDHFR1, and (B) a representative structure generated using CNS showing the secondary structure and specific loops.

 
Comparison to X-ray crystallography structure
The overall fold of the NMR and crystal (1vdr) structures are similar with an RMSD of 1.35 Å. Two different segments of the NMR structures did show a deviation from the crystal structure. First, there was a deviation in the secondary structure at the N-terminal residues of beta3 (S59–M64). The 1HN of A60 shows a strong NOE to both the 1HN of S59 and the 1HN of G58. Similarly, a strong NOE between the 1HN of S59 and the 1HN of G58 was observed in this region; however, these distances were not supported by the crystal structure. This divergence is considered to arise from the flexibility of the {alpha}2–beta3 loop. Second, there was a difference in the type of beta-turn connecting beta7 and beta8 (H147–F150). The dihedral angles of the crystal structure define a type-II beta-turn (Pieper et al. 1998) while the NMR structures define a type-I beta-turn. This type-I beta-turn is supported by the presence of an NOE from the 1HN of F150 to the 1H{alpha} of H147, which should not be observable for a type-II beta-turn. This places this turn in closer proximity to the N terminus of {alpha}1, which is supported by an NOE from the 1HN of F150 to the {delta}CH3 of I26.


   Conclusion
TOP
 Abstract
 Introduction
 Results and Discussion
 Conclusion
Materials and Methods
 Acknowledgments
 References
 
In this study we showed that a protein structure can be investigated at an extreme salt concentration by NMR spectroscopy. By taking advantage of a smaller diameter sample tube we were able to minimize the coupling between sample and coil, and succeeded in acquiring interpretable NMR data with standard NMR experimental conditions. It should also be noted that these conditions caused no sample heating problems in the spin-lock experiments. The NMR derived structure of hvDHFR1 has the same fold as the crystal structure with a 1.35 Å backbone superimposition but deviated at specific locations. The difference in the secondary structure at the N-terminal residues of beta3 causes a slight kink in the overall structure compared to the crystal structure. The adenosine binding domain has a secondary backbone superimpose of 0.83 Å with the crystal structure while the major domain is 0.66 Å. If either end of the molecule is superimposed it causes the whole other end of the molecule to deviate by as much as 2.5 Å from the crystal structure. The other deviation, which resulted in a type-I beta-turn between beta7 and beta8, may be more significant. In ecDHFR, this is an extended loop structure (G–H loop) that interacts with the M20 loop. It has been speculated that these two loops my have a correlated motion (Pan et al. 2000; Radkiewicz and Brooks 2000), and there is even a hydrogen-bond network between the G–H, M20, and F–G loops (Osborne et al. 2001). This beta-turn is too short in hvDHFR1 for a direct interaction with the corresponding L21 loop, but there may still be a correlated motion of this turn with the N terminus of {alpha}1. The type-I beta-turn of the NMR structure places this turn in a closer proximity to {alpha}1 than the type-II beta-turn of the crystal structure. Relaxation studies of ecDHFR have shown that there is a new timescale of motion for S148 in the G–H loop in the ternary ecDHFR:folate:NADP+ complex (Osborne et al. 2001). This change in motion was associated with a change in a hydrogen-bond network as the M20 loop goes from the occluded to closed conformation. It will be of interest to determine if this is present for hvDHFR1 or has the high salt eliminated the need for this interaction resulting in a smaller beta-turn.

It is most likely that it is the distribution of charges across the surface of the protein that accounts for the haloadaptation of hvDHFR1 (Bohm and Jaenicke 1994b,a). The NMR-derived structures have some shifts in the positioning of the negative side chains (D93, E95, D114, E138, and E148) compared to the crystal structure. The most dramatic change is in the position of the side chains of D93 and E148. In the NMR structures D93 is in close proximity to the side chains of D39 and D40 (~6 Å), which would create a large negative surface. The D93 side chain is pointing away from D39 and D40 (~11 Å) in the crystal structure. It has been speculated that large negative surfaces of haloproteins play a key role in haloadaptation (Bohm and Jaenicke 1994b,a). The type-I beta-turn in the NMR structures causes the side chain of E148 to come in close proximity to {alpha}1 while in the crystal structure; this residue points away from {alpha}1, which could play a role in DHF binding.

These studies set the framework for further studies to elucidate the salt-dependent activity of both hvDHFR1 and ecDHFR. The binary hvDHFR1:folate, hvDHFR1:NADPH, and ternary hvDHFR1:folate:NADPH are currently under investigation to determine if there are any conformational differences from the ecDHFR complexes. Relaxation measurements at different salt concentrations of these complexes will aid in understanding the salt-dependent flexibility which could be linked to activity, electrostatics, and halophilic adaptation.


   Materials and Methods
TOP
 Abstract
 Introduction
 Results and Discussion
 Conclusion
 Materials and Methods
Acknowledgments
 References
 
Nuclear magnetic resonance spectroscopy (NMR)
Overexpression and purification of the protein hvDHFR1 was accomplished as previously published (Binbuga and Young 2005). All NMR samples were triple labeled (2H-50%, 13C, 15N) and concentrated to 2.0 mM in 3.5 M NaCl and 10 mM Tris buffer at pH 7.0. The final sample was prepared in a 95% H2O/5% D2O mixture. NMR data was collected at 25°C using Bruker Avance 600 MHz spectrophotometer with triple resonance (1H, 13C, 15N) z-gradient probe at the Center for NMR Spectroscopy at Washington State University. The 3D 15N-NOESY-HSQC and 13C-NOESY-HSQC experiments were recorded with a mixing time of 150 msec. Raw NMR data sets were processed and analyzed using the NMRPipe software (Delaglio et al. 1995) and viewed with PIPP (Garrett et al. 1991) on a Silicon Graph Octane workstation. The chemical shift assignments have been accomplished and published (BMRB accession code: 6645) (Binbuga and Young 2005).

Structure calculation
Dihedral angle restrains {Phi} and {psi} were predicted from the program TALOS (Cornilescu et al. 1999) by using the chemical shift values of 15N, 1H{alpha}, 13C{alpha}, 13Cbeta, and 13C. The NOE distance restraints were obtained from the 15N-NOESY-HSQC and 13C-NOESY-HSQC. These data were input as experimental restraints in the crystallography and NMR system (CNS version 1.1) (Brunger et al. 1998) program to obtain NMR-derived structures. The structure calculations were continued and finalized until no NOE distance was violated by >0.5 Å, no dihedral angle violation >10°, low energies were obtained, and there was good convergence. The resultant structures were subjected to a further energy minimization due to high improper energy. A 59-step conjugated minimization was performed on each structure using the program InsightII in the Discover Module (version 2005, Accelrys). The InsightII (version 2005, Accelrys) software was used for graphical visualization. Additional graphics were prepared using MOLMOL (Koradi et al. 1996).


   Footnotes
 
Reprint requests to: John K. Young, Department of Chemistry, Mississippi State University, Box 9573, Mississippi State, MS 39762, USA; e-mail: jky1{at}ra.msstate.edu; fax: (662) 325-1618.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.072950407.


   Acknowledgments
TOP
 Abstract
 Introduction
 Results and Discussion
 Conclusion
 Materials and Methods
 Acknowledgments
References
 
We thank Dr. Lisa M. Gloss from Washington State University for supplying us with the plasmid containing the hvDHFR1 gene and Dr. Gregory L. Helms for collecting the NMR data.


   References
TOP
 Abstract
 Introduction
 Results and Discussion
 Conclusion
 Materials and Methods
 Acknowledgments
 References
 
Binbuga, B. and Young, J.K. 2005. 1H, 13C, and 15N backbone and side chain resonance assignments of Haloferax volcanii DHFR1. J. Biomol. NMR 33: 281.[Medline]

Boehr, D.D., McElheny, D., Dyson, H.J., and Wright, P.E. 2006. The dynamic energy landscape of dihydrofolate reductase catalysis. Science 313: 1638–1642.[Abstract/Free Full Text]

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Bohm, G. and Jaenicke, R. 1994a. Relevance of sequence statistics for the properties of extremophilic proteins. Int. J. Pept. Protein Res. 43: 97–106.[Medline]

Bohm, G. and Jaenicke, R. 1994b. A structure-based model for the halophilic adaptation of dihydrofolate reductase from Halobacterium volcanii . Protein Eng. 7: 213–220.[Abstract/Free Full Text]

Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., et al. 1998. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54: 905–921.[CrossRef][Medline]

Bystroff, C. and Kraut, J. 1991. Crystal structure of unliganded Escherichia coli dihydrofolate reductase. Ligand-induced conformational changes and cooperativity in binding. Biochemistry 30: 2227–2239.[CrossRef][Medline]

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Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J., and Bax, A. 1995. NMRPipe: A multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6: 277–293.[Medline]

Falzone, C.J., Wright, P.E., and Benkovic, S.J. 1994. Dynamics of a flexible loop in dihydrofolate reductase from Escherichia coli and its implication for catalysis. Biochemistry 33: 439–442.[CrossRef][Medline]

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Laskowski, R.A., MacArthur, M.W., Moss, D.S., and Thornton, J.M. 1993. PROCHECK: A program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26: 283–291.[CrossRef]

Lee, H., Reyes, V.M., and Kraut, J. 1996. Crystal structures of Escherichia coli dihydrofolate reductase complexed with 5-formyltetrahydrofolate (folinic acid) in two space groups: Evidence for enolization of pteridine O4. Biochemistry 35: 7012–7020.[CrossRef][Medline]

Marg, B.L., Schweimer, K., Sticht, H., and Oesterhelt, D. 2005. A two-alpha-helix extra domain mediates the halophilic character of a plant-type ferredoxin from halophilic archaea. Biochemistry 44: 29–39.[CrossRef][Medline]

McElheny, D., Schnell, J.R., Lansing, J.C., Dyson, H.J., and Wright, P.E. 2005. Defining the role of active-site loop fluctuations in dihydrofolate reductase catalysis. Proc. Natl. Acad. Sci. 102: 5032–5037.[Abstract/Free Full Text]

Mevarech, M., Frolow, F., and Gloss, L.M. 2000. Halophilic enzymes: Proteins with a grain of salt. Biophys. Chem. 86: 155–164.[CrossRef][Medline]

Osborne, M.J., Schnell, J., Benkovic, S.J., Dyson, H.J., and Wright, P.E. 2001. Backbone dynamics in dihydrofolate reductase complexes: Role of loop flexibility in the catalytic mechanism. Biochemistry 40: 9846–9859.[CrossRef][Medline]

Pan, H., Lee, J.C., and Hilser, V.J. 2000. Binding sites in Escherichia coli dihydrofolate reductase communicate by modulating the conformational ensemble. Proc. Natl. Acad. Sci. 97: 12020–12025.[Abstract/Free Full Text]

Pieper, U., Kapadia, G., Mevarech, M., and Herzberg, O. 1998. Structural features of halophilicity derived from the crystal structure of dihydrofolate reductase from the Dead Sea halophilic archaeon, Haloferax volcanii . Structure 6: 75–88.[Medline]

Pundak, S., Aloni, H., and Eisenberg, H. 1981. Structure and activity of malate dehydrogenase from the extreme halophilic bacteria of the Dead Sea. 2. Inactivation, dissociation and unfolding at NaCl concentrations below 2 M. Salt, salt concentration and temperature dependence of enzyme stability. Eur. J. Biochem. 118: 471–477.[Medline]

Radkiewicz, J.L. and Brooks, C.L.I. 2000. Protein dynamics in enzymatic catalysis: Exploration of dihydrofolate reductase. J. Am. Chem. Soc. 122: 225–231.[CrossRef]

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