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Electrostatic interactions in neurotoxin II from Asian cobra Naja oxiana

Neurotoxin and acetylcholine receptor

Snake’s neurotoxins are relatively small proteins containing 60-75 amino acid residues. Their biological function is blocking of neuro-muscular transmission. The target for neurotoxins is nicotinic acetylcholine receptor (nAChR) located on the membranes of muscular cells in sinaps. Neurotoxins bind to nAChR (Fig.1) thus screening the receptor active sites.

Fig. 1 Scheme of nAChR - neurotoxin complex.

Neurotoxins represent a powerful tool to study nAChR functioning. 3D structures of some neurotoxins were determined using X-ray and NMR data. Mutagenesis experiments show that Arg and Lys residues located on the top of protein loops are most important for binding to the receptor. Interestingly, small differences in the neurotoxins sequences may strongly affect their affinity to the receptor. To study the structure-function relationship of a protein it is important to explore its conformation and dynamical behavior with experimental and molecular modeling methods.

The project is devoted to theoretical studies of structure and dynamics of the neurotoxin-II Naja oxiana (NTII). A set of high-resolution 3D structures of NTII in aqueous solution has been obtained in our lab using NMR spectroscopy [1]. However these models do not represent completely the real conformational space of the molecule, and some refinement is needed. We used molecular dynamics (MD) method[2] to explore the conformational flexibility of NTII. Also, new information was extracted from NMR spectra of NTII in water at different pH. Analysis of chemical shifts of 1H signals as a function of pH (d(pH)) and MD results help to refine NMR-derived 3D structure of NTII.

Measurement of pKa values and identification of h-bonds:

pKa values of ionizable groups of Asp, Glu and His in NTII (Table 1) were estimated based on d(pH) dependences obtained from the analysis of 1H NOESY spectra. pKa’s of groups in denaturated proteins (where these groups are fully solvated) were taken as reference.

Table 1 pKa values of ionizable groups of NTII

Glu23.8 (4.3)
Asp572.7 (4.0)
C-terminal1.2## (3.8)
# from 1H TOCSY NMR spectra at pH 6.5-12.0.
## from optical spectroscopy.

Several transient H-bonds in NTII were identified in analysis of d(pH) of backbone NH with carboxyl groups: Glu2:NH - Od:Asp57; Ser18:NH, Glu20:NH, Asn22:NH - Oe: Glu20; Arg32:NH - Od:Asp30 (Fig.2). Interestingly, d(pH) for the NH group of Thr14 (pKa 4.9 is assigned to His4) shows abnormally high amplitude. This is the effect of the H-bond His4 NHd1 - O Thr13 which agrees well with the NMR-derived constraints on the side chain of His4.

Fig. 2 Identification of H-bonds in NTII based on analysis of the d(pH) dependencies. NTII is shown in ribbon respresentation. Side chains of Asp and Glu residues are colored in magenta, Arg and Lys side chains - in blue. His4 and His31 are electroneutral at pH 7. Some high amplitude d(pH) dependencies of NH are shown.

Molecular dynamics study of NTII

Conformation space of NTII in aqueous solution was explored via 2 ns MD simulations. MD results were tested against the experimental data - NOE constraints and H-bonds stability.

MD simulations show that the conformational change in loop I leads to rearrangement of its interface with loop II (Fig.3). This results in NOE constraints violations and modification of the H-bonds set. The key event in this transition is rotation of the Gln6:NH group and formation of H-bonds Gln6 NH - O Arg38.

Fig.3 Hydrogen bonds in the MD structure of NTII. Interface between loops I and II of NTII in one of MD-derived conformation. Main chain of the loop I is colored in green , loop II - in yellow, - C-terminal residues 56-61 - in violet.

The obtained curves d(pH), along with MD data, show that the side chain of Gln6 is stable and may form H-bonds Gln6 NH - Oe1 Gln6 and Thr13 NH - Oe1 Gln6 (these H-bonds have not been observed in the NMR structures of NTII). MD simulations with weak constraints on these H-bonds demonstrate stabilization effects for them. The interface between loops I and II , conformation of loop I and C-terminal residues remain stable in MD. The following network of H-bonds which stabilize the conformation of NTII proposed based on MD data: Asn60 Hd21 - O Tyr24, Asn60 Hd22 - O His4, Asn60 NH - Od1 Asn60, His4 Hd1 - O Thr13, Gln6 NHe21 - Od1 Asn61.

Based on the analysis of pH-dependent chemical shifts in 1H NMR spectra and MD simulations we refine the H-bonds network in NTII. These H-bonds play important role in stabilization of the native structure. In future we plan to develop MD protocol for more correct reproducing the dynamical behavior of neurotoxins in water. Study of conformational flexibility of neurotoxins is indispensable to uderstand their structure-function relationship.


  1. Golovanov A. P. et al. Eur. J. Biochem. 213, 1213–1223 (1993);
  2. The GROMACS software (www.gromacs.org);
  3. Szyperski T. et al. Biochemestry. 33, 9303–9310 (1994).

(Authors: Yuri Kosinsky, Alexander Arseniev, Roman Efremov).

Address: 117997 Russia, Moscow, ul. Miklukho-Maklaya 16/10.
Tel.: +7 (495) 336-20-00.
Email: efremov@nmr.ru

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