Dimerization of Nitrophorin 4 at Low pH and Comparison to the K1A Mutant of Nitrophorin 1

The nitrophorins (nitro = NO, phorin = carrier) make up a group of NO-carrying heme proteins found in the saliva of at least two species of blood-sucking insects, Rhodnius prolixus, the “kissing bug”, which has four such proteins in the adult insect1−5 and at least three additional nitrophorins in earlier stages of development,6,7 and Cimex lectularius, the bedbug, which has only one.8,9 These interesting heme proteins sequester NO that is produced by a nitric oxide synthase (NOS) that is similar to vertebrate constitutive NOS and is present in the endothelial cells of the salivary glands,10−12 which keeps it stable for long periods of time by binding it as an axial ligand to a ferriheme center.1,3−5 The nitrophorins are at a very high concentration in the salivary glands of R. prolixus (combined NP concentration estimated to be ∼6–10 mMa), thus giving rise to the cherry red color1 of the glands. To function in insect feeding, the nitrophorin proteins must efficiently pack in the gland and stably bind NO, despite its reactive nature. The ferriheme binding site is crucially important in stabilizing the bound NO for long periods of time in the salivary glands. Upon injection into the tissues of the victim, NO dissociates and diffuses through the tissues to the nearby capillaries to cause vasodilation and thereby allow more blood to be transported to the site of the wound. At the same time, histamine, whose role is to cause swelling, itching, and the beginning of the immune response, is released by mast cells and platelets of the victim in the region of the bite. In the case of the Rhodnius proteins, this histamine binds to the heme sites of the nitrophorins, hence preventing the insect’s detection for a period of time.13 These two properties of the nitrophorins of R. prolixus contribute to the transmission of the protozoan Trypanasoma cruzi, the vector of Chagas’ disease,14 to the victim, via the feces of the insect, which are left behind at the site of the bite3 following the extended feeding time. The Rhodnius nitrophorins of the adult insect, which have been named NP1–NP4 in reverse order of their abundance in the saliva,2 occur as two pairs of similar sequence proteins, NP1 and NP4, which are 90% identical, and NP2 and NP3, which are 80% identical;2 the overall level of sequence identity is only 38%. The sequences are shown in Figure S1 of the Supporting Information. These proteins have been investigated by a number of techniques (DOI: 10.1021/bi501305a),1,3,15−41 and the solid state structures of one or more ligand complexes of NP1,15,42 NP2,43,44 and NP445−50 have been determined by X-ray crystallography. The structures are unique for heme proteins, in that the heme is located inside, but at the open end, of a β-barrel,8,51 as shown in Figure ​Figure1,1, rather than in the more commonly observed largely α-helical globin52 or four-helix bundle53 folds. The ferriheme molecule is bound to the protein via a histidine ligand, and the sixth coordination site is available to bind NO or other ligands. In the NO-off form in vitro, either water or ammonia, depending on buffer type, is bound to the sixth site.42,45 Figure 1 Structure of NP4. Shown are the protein backbone (blue for β-strands, red for α-helices, and gray for loops) and the heme (gold). Taken from Protein Data Bank entry 1X8O. Although NP4 and quite a number of its axial ligand complexes have been crystallized and their structures determined to high resolution by single-crystal X-ray diffraction,45−50 NP4 in solution at the low pH of the insect’s saliva (5–6) is an equilibrium mixture of at least two forms, a monomer and a dimer; higher-order oligomers have also been claimed.54 The previous report focused mainly on the gas-phase properties of NP4, which showed up to 14-mers present by mass spectrometry.54 The work presented here focuses on the solution properties that are important to the reaction chemistry of NP4 and its NO complex in the salivary glands and the tissues of the victim. Our work has included the preparation and investigation of site-directed mutants to define which protein side chains are involved in dimerization. We find that NP4 is a dimer at pH 5.0 at ≥1 mM but a monomer at pH 7.3, the approximate pH of the victim’s tissues; the dimer is much more stable when the ferriheme iron is bound to nitric oxide (NO). Because the sequences of NP4 and NP1 are 90% identical, we were surprised that we had not observed a dimeric form for NP1 during our early studies of that protein.15,17,19,21,23,25,28 However, NP1, like NP2 and NP3, has a charged amino acid at its N-terminus (Figure S1 of the Supporting Information), and during expression of any of these three genes, the methionine that results from translation of the start codon of the gene is not cleaved by the methionine aminopeptidase of Escherichia coli, thus leaving M0, with its relatively large side chain, at the N-terminus of these three proteins when they are isolated and purified. In the insect, the nitrophorins are expressed with an N-terminal signal sequence to target the protein for secretion into the salivary gland of the insect; cleavage of the signal sequence after secretion yields the mature N-terminus without methionine. The genes for the recombinant proteins, however, did not include the N-terminal signal sequence but rather began with the start codon, followed by the codon for the first amino acid of the protein. The M0 residue in the recombinant protein was not shown in the crystal structures of NP1 published before 2000,15,42 because of the relatively low resolution of the structures (2.0–2.3 A) and disorder at the N-terminus in the crystals, but mass spectrometry clearly shows the presence of M0 for recombinant NP1, as well as NP224 and NP3, as shown below. In contrast to NP1–NP3, NP4, with its N-terminal amino acid alanine, does not retain M0 when expressed recombinantly. We thus suspected that because NP1 [actually (M0)NP1] did not form a dimer, while NP4 did, the N-terminal region of the NP4 and NP1 proteins might be involved in the observed dimerization of NP4, and we have thus prepared the gene for the K1A mutant of NP1 as well as native N-terminal NP1 in this work. Our precedent for this approach was our earlier work on the D1A mutant of NP2,24 which we found had no M0 present when isolated and had properties markedly different from those of the (M0)NP2 obtained from simple expression of the recombinant gene.24 Later, we also prepared native N-terminal NP2, with D1 as the first amino acid, by combining our NP2 gene with an export sequence provided by the pelB leader sequence from Erwinia carotovora, which is present in the pET-26b expression plasmid (Novagen), to export the protein to the periplasm.29 When the export sequence was naturally cleaved in the E. coli periplasm, we were left with native N-terminal NP2, which could be purified in small quantities. The expression also had to be conducted in small batches, and thus, we have continued to use NP2(D1A) for experiments that require large quantities of protein. We found that these two proteins [NP2(D1A) and native N-terminal NP2] had essentially identical heme 1H NMR spectra and 1H{15N} HSQC spectra and very similar reaction properties, except for the rate constant for NO binding at pH 7.5, which was a factor of 5 smaller, and the equilibrium Kd for NO, which was a factor of 5 larger for the native N-terminal NP2 than for NP2(D1A).29 These findings suggest an important role for D1 in the reactivity of the protein,29 which we have not as yet elucidated. We have likewise recently created N-terminal NP1 in the same manner but find again that NP1(K1A) is easier to prepare in large quantities. We have found that the techniques of size-exclusion chromatography at pH 5.0 and multidimensional NMR spectroscopy over a range of pH values from 4.0 to 8.0 are the most useful for studying the dimer/monomer behavior of NP4 and its mutants, as well as NP1(K1A) and native N-terminal NP1 in solution, and we report our findings below.