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Effect of the N-terminus on heme cavity structure, ligand equilibrium, rate constants, and reduction potentials of nitrophorin 2 from Rhodnius prolixus.

ABSTRACT: The D1A mutant of recombinant NP2 has been prepared and shown to have the expression-initiation methionine-0 cleaved during expression in E. coli, as is the case for recombinant NP4, where Ala is the first amino acid for the recombinant protein as well as for the mature native protein. The heme substituent 1H NMR chemical shifts of NP2-D1A and those of its imidazole, N-methylimidazole, and cyanide complexes are rather different from those of NP2-M0D1. This difference is likely due to the much smaller size of the N-terminal amino acid (A) of NP2-D1A, which allows the formation of the closed loop form of this protein, as it does for NP4 (Weichsel, A., Andersen, J. F., Roberts, S. A., and Montfort, W. R. (2000) Nature Struct. Biol. 7, 551-554). The ratio of the two hemin rotational isomers A and B is different for the two proteins, and the rate at which the A:B ratio reaches equilibrium is strikingly different (NP2-M0D1 t1/2 for heme rotation approximately 2 h, NP2-D1A t1/2 approximately 43 h). This difference is consistent with the high stability of the closed loop form of the NP2-D1A protein and infrequent opening of the loops that could allow heme to at least partially exit the binding pocket in order to rotate about its alpha,gamma-meso axis. Consistent with this, the rates of histamine binding and release to/from NP2-D1A are significantly slower than those for NP2-M0D1 at pH 7.5. This work suggests that care must be taken in interpreting data obtained from proteins that carry the expression-initiation M0.

SUBMITTER: Berry RE

PROVIDER: S-EPMC2518688 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Effect of the N-terminus on heme cavity structure, ligand equilibrium, rate constants, and reduction potentials of nitrophorin 2 from Rhodnius prolixus.

Berry Robert E RE Shokhireva Tatiana Kh TKh Filippov Igor I Shokhirev Maxim N MN Zhang Hongjun H Walker F Ann FA

Biochemistry 20070517 23

The D1A mutant of recombinant NP2 has been prepared and shown to have the expression-initiation methionine-0 cleaved during expression in E. coli, as is the case for recombinant NP4, where Ala is the first amino acid for the recombinant protein as well as for the mature native protein. The heme substituent 1H NMR chemical shifts of NP2-D1A and those of its imidazole, N-methylimidazole, and cyanide complexes are rather different from those of NP2-M0D1. This difference is likely due to the much sm ...[more]

PMID: 17506528

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Project description:Nitrophorins (NPs) are a class of NO-transporting and histamine-sequestering heme b proteins that occur in the saliva of the bloodsucking insect Rhodnius prolixus. A detailed study of the newly described member, NP7, is presented herein. NO association constants for NP7 [KIII(eq)(NO)] reveal a drastic change when the pH is varied from 5.5 (reflecting the insect's saliva) to slightly above plasma pH (7.5) (>10(9) M-1 --> 4.0 x 10(6) M-1); thus, the protein promotes the storage of NO in the insect's saliva and its release inside the victim's tissues. In contrast to the other nitrophorins, NP1-4, histamine sequestering cannot be accomplished in vivo due to the low binding constant [KIII(eq)(histamine)] of 10(5) M-1 compared to the histamine concentration of 1-10 x 10(-9) M in the blood. A major part of this study deals with the N-terminus, 1Leu-Pro-Gly-Glu-Cys5 of NP7, which is not found in NP1-4. Since NP7 has not been isolated from the insects but was recognized in a cDNA library instead, the N-terminal site of signal peptidase cleavage upon protein secretion was predicted by the program SIGNALP [Andersen, J. F., Gudderra, N. P., Francischetti, I. M. B., Valenzuela, J. G., and Ribeiro, J. M. C. (2004) Biochemistry 43, 6987-6994]. In marked contrast to wild-type NP7, NP7(Delta1-3) exhibits a very high NO affinity at pH 7.5 [KIII(eq)(NO) approximately 10(9) M-1], suggesting that the release of NO in the plasma cannot efficiently be accomplished by the truncated form. Comparison of the reduction potentials of both constructs by spectroelectrochemistry revealed an average increase of +85 mV for various distal ligands bound to the heme iron when the 1Leu-Pro-Gly3 peptide was removed. However, 1H NMR and EPR spectroscopy show that the electronic properties of the FeIII cofactor are similar in both wild-type NP7 and NP7(Delta1-3). Further, thermal denaturation that revealed a higher stability of wild-type NP7 compared to NP7(Delta1-3), in combination with a homology model based on the NP2 crystal structure (rmsd = 0.39 A), suggests that interaction of the 1Leu-Pro-Gly3 peptide with the A-B and/or G-H loops is key for proper protein function.

Dataset Information

Effect of the N-terminus on heme cavity structure, ligand equilibrium, rate constants, and reduction potentials of nitrophorin 2 from Rhodnius prolixus.

Publications

Effect of the N-terminus on heme cavity structure, ligand equilibrium, rate constants, and reduction potentials of nitrophorin 2 from Rhodnius prolixus.

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