Solution structure of acyl carrier protein from <i>Nitrosomonas europaea</i>

Here, we report the solution structure of Q82SY3 from Nitrosomonas europaea. N. europaea is a Gram-negative bacterium with the interesting capability to derive all its energy from oxidation of ammonia to nitrite. It lives in several places such as soil, sewage, fresh water, walls of buildings, and on the surface of monuments, especially in polluted areas where air contains high levels of nitrogen compounds. N. europaea and other ammonia-oxidizing bacteria play a very crucial role in the biogeochemical nitrogen fixing cycle, that is, biological conversion of reduced nitrogen in the form of ammonia or ammonium to oxidized nitrogen in the form of nitrite, nitrate, or gaseous forms (NO, N2O), which in turn, enhances nitrogen availability to plants and waste water1 treatment. We believe Q82SY3 is likely to be an acyl carrier protein (ACP) based on its structural similarity with such proteins from other organisms such as Escherichia coli and the presence of a conserved “DS” motif. In Figure 1(A), the conserved “DS” motif is highlighted in the sequence alignment of acyl carrier proteins from various sources. ACP is a small protein family involved in fatty acid biosynthesis. ACP is a cytosolic transport protein that carries fatty acid via thioester linkage facilitated by the conserved serine residue for chain elongation by fatty acid synthase system.2, 3 In addition to its function as a carrier protein, ACP could also modulate activities of the enzymatic system involved in fatty acid synthesis. Evidence suggests that substituting ACP from one organism to another yields significant variation in the fatty acid distribution.4 Involvement of ACPs in fatty acid biosynthesis and antibiotic biosynthesis warrants the study of the structure–function relationships of this class of proteins. (A) Sequence alignment of Q82SY3 (top row) with other acyl carrier proteins (ACP) from various organisms. The secondary structural elements (α-helices) of Q82SY3 have been included just above the sequence. The conserved catalytic site “DS” has been shown underlined with a bold font. The residues are colored as follows: red: hydrophobic/aromatic; blue: acidic; magenta: basic; green: all others. (B) Stereoview of the backbone alignment of an ensemble of 20 structures of Q82SY3. N- and C-terminals are indicated and helices are color coded as helix I—red; helix II—green and helix III—yellow. Q82SY3 was cloned in p11 vector using recombinant technology and expressed in E. coli BL21-DE3 (gold) cells with an N-terminal His-tag of sequence “MGSSHHHHHHSSGRENLYFQGH” to facilitate protein purification. Bacterial cultures were grown in 37°C to an OD600 of 0.6 and induced with 1 mM IPTG for overnight at 15°C. The protein was purified to homogeneity using metal (Ni) affinity chromatography. His tag was not cleaved off from the protein sequence. Uniformly 15N and 13C labeled protein samples were prepared in M9 media supplemented externally with 15N ammonium chloride (1 g/L) and 13C6-glucose (2 g/L). The NMR sample was prepared in 95% H2O/5% D2O mixture using 10 mM MOPS buffer (pH = 6.5), 450 mM NaCl, 10 mM DTT, 1 mM Benzamidine, and 0.01% NaN3. All NMR spectra were collected at 25°C on a Bruker Avance 600 MHz spectrometer equipped with cyro probe. The program NMRPIPE5 was used to process all NMR data with linear prediction to improve digital resolution [on indirect dimensions] and peak picking was done with Sparky.6 The complete backbone assignments were achieved using CBCA(CO)NH, HNCA, HNCB, HBHA(CO)NH, HNCO and HN(CO)CA spectra and assisted by the use of the program Monte.7 The side chain assignments were done manually using CC(CO)NH-Tocsy, HCC(CO)NH-TOCSY, HC(C)H-TOCSY and HCC(H)-TOCSY spectra. All together, 95.7% of assignment was completed and deposited in BMRB (entry id. 6769). Automated NOE assignments and structure calculation were carried out using CANDID module of Cyana 2.08 using peak lists derived from a time-shared (13C, 15N) NOESY-HSQC (mixing time = 150 ms).9 The CYANA-assigned NOEs were confirmed manually. Only about 12% of peaks were left unassigned due to ambiguity. The structure ensemble calculated by CYANA did not determine a unique assignment for such NOESY peaks, based on chemical shift assignments and tolerances. Also, 43 stereospecific assignments (37 methylene, 5 methyl, and 1 side-chain amide protons) were defined by CYANA2.0. The backbone dihedral angles were obtained from TALOS10 and were used as angle restraints in calculation. Similarly, hydrogen bond constraints were derived from slowly exchanging amide protons indicated by 15N-HSQC recorded after a day of room temperature incubation of Q82SY3 exchanged with deuterated buffer. From a total of 100 structures, 20 structures were selected with the lowest CYANA target function, for refinement using CNS. The upper limit distant restraints, upper limit hydrogen bond constraints, and angle constraints from CYANA were converted into CNS format and used along with RDC restraints for refinement in the presence of explicit water.11 The RMSD of the ensemble was calculated using MOLMOL.12 To improve the quality of the NMR structure, residual dipolar couplings were measured by aligning Q82SY3 in filamentous phage (Pf1) medium (purchased from ASLA Ltd. Riga Latvia). The basic RDC sample preparation protocol was adopted from Zweckstetter et al.,13 but the final concentration of Pf1 was kept at 12 mg/mL. The RDC measurement on partially aligned Q82SY3 was done by collecting a series of 1JNH-modulated HSQC13 specra. The program PALES15 was used for the analysis of RDC data. Twenty best structures were selected from a pool of 100 structures. The structures were refined with distance, angle, and RDC restraints. The calculated order parameters from RDC data and structural statistics are given in Table I. Superposition of backbone of 20 refined conformers of Q82SY3 is depicted in Figure 1(B). The solution structure of Q82SY3 has the three helix bundle motif of ACP. Helix I runs from residues 3 to 15, helix II from 40 to 52, and helix III from 71 to 81. Relatively long loops connect these helices with one another. The length of these loops may vary between different organisms. Comparison of solution structures of Q82SY3 with two other ACPs from E. coli and Lactobacillius casei highlights the structural similarities and supports the hypothesis on possible functions of Q82SY3 [Fig. 2(A)]. The transport of fatty acids by ACP has been achieved through esterification of serine residues. This necessitates the exposure of serine residues and the presence of nearby hydrophobic patches to facilitate fatty acid binding. On comparing the sequence of ACPs from other organisms it is found that the “DS” motif has been conserved invariably. In Q82SY3, Asp38 and Ser39 form the esterification catalytic active site. As seen in Figure 2(B), Asp38 and Ser39 are present as exposed residues in the loop just above helix II. Also, in Figure 2(B), a hydrophobic cleft is clearly visible between helix II and helix III, and is partly contributed by the loop connecting these two helices. This cleft consists of the following residues: Val42, Ile46, and Leu49 from helix II, Leu73 and Ala74 of helix III, Phe67, Ile62, and Ala64 of loop between helix II and helix III, and Leu31 of the loop between helix I and helix II. So, the conserved active residues (Asp38 and Ser39) and the nearby hydrophobic cleft aid Q82SY3 to serve as a fatty acid carrier protein in fatty acid synthase system. DALI,16 a structural homology server, predicted Q82SY3 is structurally homologous to D-Alanyl carrier protein (Dcp). The Z-score of this prediction was 7.9, and the percentage of identity was 22%. The D-alanylation of Lipoteichoic acid (LTA) modulates the surface charges of bacteria, thereby accounting for the electrochemical properties of the cell wall.17 The D-analylation to LTA requires D-alanine:D-alanyl carrier protein ligase (Dcl) and D-alanyl carrier protein (Dcp).17 A significant secondary structure similarity is observed upon comparing structures of Q82SY3 and Dcp (pdb 1HQB).17 Superposition of secondary structures (only residues within helices are considered for superposition) of these two proteins yield RMSD of 2.5 Å. Although the presence of the active site (Asp38 and Ser39) and nearby hydrophobic cleft facilitate Dcl ligation of D-alanine to Dcp, the transfer of D-alanine to LTA could be hindered because of the absence of the “RKEWD” motif in Q82SY3. But the “RKEWD” motif is found in all Dcps. Hence, we hypothesize that Q82SY3 might be an acyl carrier protein involved in lipid metabolism. (A) Structural comparison of Q82SY3 with two other acyl carrier proteins from PDB. The name of the organism and the PDB access code are given for each structure. (B) A surface diagram of Q82SY3 depicting conserved serine (Asp38 and Ser39) and the residues involve in forming a hydrophobic cleft (Val42, Ile46, and Leu49 from helix II, Leu73 helix III, and Phe67 of the loop between helix II and helix III in the sphere model). Only a few residues are labeled for clarity. The chemical shifts have been submitted to BMRB (accession #6769), and the structure ensemble has been deposited in PDB (accession #2AMW). C.H.A. holds a Canada Research Chair from the Canadian Institutes of Health Research. The authors thank Drs. Alexander Lemak and Bin Wu for helpful comments and suggestions throughout this project. We thank our protein production team in Toronto (http://www.uhnres.utoronto.ca/proteomics/) for cloning, expression, and purification. We thank the staff of Bruker Canada for their help with pulse programs and spectrometer maintenance. This research was supported by the Ontario Research and Development Challenge Fund; Genome Canada; the Protein Structure Initiative of the National Institutes of Health (Grant: P50-GM62413) through the Northeast Structural Genomics Consortium.

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.