Electrostatic interactions in neurotoxin II from Asian cobra Naja oxiana
Neurotoxin and acetylcholine receptor
Snakes 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.
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.
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. pKas of groups in denaturated proteins (where these groups are fully solvated) were taken as reference.
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.
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.
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.
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