NMR studies of the dynamics of nitrophorin 2 bound to nitric oxide

Abstract

The Rhodnius nitrophorins are β-barrel proteins of the lipocalin fold with a heme protruding from the open end of the barrel. They are found in the saliva of the blood-sucking insect Rhodnius prolixus, which synthesizes and stores nitric oxide (NO) in the salivary glands, where NO is bound to iron. NO is released by dilution and an increase in pH when the insect spits its saliva into the tissues of a victim, to aid in obtaining a blood meal. In the adult insect, there are four nitrophorins, NP1-NP4. At pH 7.3, NP4 releases NO 17 times faster than NP2 does, as measured by stopped-flow kinetics. A number of crystal structures of the least abundant protein, NP4, are available. These structures have been used to propose that two loops between adjacent β-strands at the front opening of the protein, the A-B and G-H loops, determine the rate of NO release. To learn how the protein loops contribute to the release of NO for each of the nitrophorins, the dynamics of these proteins are being studied in our laboratory. In this work, the NP2-NO complex has been investigated by nuclear magnetic resonance relaxation measurements to probe the picosecond-to-nanosecond and microsecond-to-millisecond time scale motions at three pH values, 5.0, 6.5, and 7.3. It is found that at pH 5.0 and 6.5, the NP2-NO complex is rigid and only a few residues in the loop regions show dynamics, while at pH 7.3, somewhat more dynamics, particularly of the A-B loop, are observed. Comparison to other lipocalins shows that all are relatively rigid, and that the dynamics of lipocalins in general are much more subtle than those of mainly α-helical proteins.

Figure 1

Structure of NP2 and pattern of connections of disulfide bonds.

Figure 2

a) Comparison of pH 5.0 (red) and 7.3 (blue) ¹H{¹⁵N} HSQC plots for NP2-NO at 30 °C at 600 MHz ¹H frequency. b) Comparison of pH 5.0 (red) and 6.5 (green) HSQC plots for NP2-NO at 30 °C at 600 MHz ¹H frequency. Note the much greater similarity of the chemical shifts of most residues at the two pH values than seen in a).

Figure 3

a) Closeup of a comparison of the pH 5.0 and 7.3 HSQC plots for NP2-NO at 30 °C, recorded at 600 MHz, showing the dramatic change in the chemical shifts of several residues. b) Absolute change in ¹⁵N chemical shift between these two pH values for the most-shifted residues (those of the A-B and G-H loops).

Figure 4

¹⁵N relaxation data and calculated modelfree order parameter (S²) of the native N-terminus NP2-NO complex measured at pH 5.0. The modelfree order parameters were obtained by fitting the raw data to the extended Lipari-Szabo modelfree formalism using FASTModelfree as described in the Materials and Methods. Errors are not shown for clarity. The location of β-sheets and helices are shown above the plot. A total of 29 non-proline residues could not be included in the calculation either because of overlap (27 residues) or not being assigned (2 residues).

Figure 5

¹⁵N relaxation data and calculated modelfree order parameter (S²) of the native N-terminus NP2-NO complex measured at pH 7.3. The modelfree order parameters were obtained by fitting the raw data to the extended Lipari-Szabo modelfree formalism using FAST-Modelfree as described in the Materials and Methods. Errors are not shown for clarity. The location of β-sheets and helices are shown above the plot. A total of 30 non-proline residues could not be included in the calculation for various reasons such as signal overlap (15 residues), very weak signal intensity (7 residues) and not being assigned (8 residues).

Figure 6

Comparison of the modelfree data for R₁, R₂ and ¹H{¹⁵N} NOE as a function of pH for the A-B and G-H loops, showing that R₁ is not affected, while R₂ and ¹H{¹⁵N} NOE are significantly affected by the change in pH. This behavior of the R₂ values is consistent with the fact that the motions of the A-B loop in particular are on a much slower timescale than pico- to nanoseconds which are probed by the modelfree experiments.

Figure 7

Per residue plot of ΔR₂^eff(ν_CPMG) for the native N-terminus NP2-NO complex, showing motions in the micro- to millisecond timescale, at three different pH values (5.0, 6.5 and 7.3).