Báo cáo khoa học: The effect of replacing the axial methionine ligand with a lysine residue in cytochrome c-550 from Paracoccus versutus assessed by X-ray crystallography and unfolding

The Effect Of Replacing The Axial Methionine Ligand With A Lysine Residue In Cytochrome C-550 From Paracoccus Versutus Assessed By X-ray Crystallography And Unfolding

Tác giả: Jonathan A. R. Worrall, Anne-Marie M. van Roon, Marcellus Ubbink and Gerard W. Canters

Lĩnh vực: Hóa sinh, Sinh học cấu trúc

Nội dung tài liệu: Nghiên cứu này trình bày kết quả phân tích cấu trúc tinh thể tia X và quá trình biến tính của cytochrome c-550 từ vi khuẩn Paracoccus versutus. Cụ thể, bài báo tập trung vào ảnh hưởng của việc thay thế gốc methionine (Met) ở vị trí phối trí trục thứ sáu bằng gốc lysine (Lys) lên cấu trúc ba chiều, sự ổn định và hoạt tính của protein. Các phương pháp chính được sử dụng bao gồm nhiễu xạ tia X, phổ UV-Vis và cộng hưởng từ hạt nhân (NMR).

Mục lục chi tiết:

• Abstract
• Keywords
• Correspondence
• (Received 14 January 2005, revised 9 March 2005, accepted 15 March 2005)
• doi:10.1111/j.1742-4658.2005.04664.x
• The structure of cytochrome c-550 from the nonphotosynthetic bacteria Paraccocus versutus has been solved by X-ray crystallography to 1.90 Å resolution, and reveals a high structural homology to other bacterial cytochromes c2.
• The effect of replacing the axial heme-iron methionine ligand with a lysine residue on protein structure and unfolding has been assessed using the M100K variant.
• From X-ray structures at 1.95 and 1.55 Å resolution it became clear that the amino group of the lysine side chain coordinates to the heme-iron.
• Structural differences compared to the wild-type protein are confined to the lysine ligand loop connecting helices four and five.
• In the heme cavity an additional water molecule is found which participates in an H-bonding interaction with the lysine ligand.
• Under cryo-conditions extra electron density in the lysine ligand loop is revealed, leading to residues K97 to T101 being modeled with a double main-chain conformation.
• Upon unfolding, dissociation of the lysine ligand from the heme-iron is shown to be pH dependent, with NMR data consistent with the occurrence of a ligand exchange mechanism similar to that seen for the wild-type protein.
• The Gram-negative, nonphotosynthetic bacterium Paracoccus versutus (formerly Thiobacillus versutus [1]) has a repertoire of respiratory chains and regulatory systems that allow it to cope with a variety of environments and different substrates as energy sources [2].
• Regardless of which energy source is utilized, a class I c-type cytochrome, cyt c-550, acting as an electron-carrier is present in the respiratory chain which is homologous to the cyts c2 found in photosynthetic bacteria [2,3].
• A number of biochemical and biophysical studies on P. versutus cyt c-550 have been reported [4-14].
• However, throughout these studies no 3D structure of the protein was available and a model based on the known structure of the cyt c-550 from P. dentirificans [15] was constructed to aid in interpretation of data.
• All class I c-type cyts so far studied, undergo a dynamic equilibrium process in their ferric state which involves the dissociation of the weak methionine-S³-heme-iron bond.
• Such a process results in an equilibrium between low- and high-spin heme species and allows for exogenous ligands such as imidazole to bind the heme-iron [16,17].
• This equilibrium also leads to cyts c possessing residual peroxidase activity under native conditions [18].
• For exogenous ligands to gain access to the heme, sufficient movement of the loop containing the Met ligand connecting helices four and five must occur [19-22].
• At present two X-ray structures of cyts c2 exist in which the coordinating Met-iron bond is broken and the vacant heme coordination site is filled by an exogenous ligand; the imidazole adduct of Rhodobacter sphaeroides cyt c2 [23] and the ammonia adduct of Rhodopseudomonas palustris cyt c2 [24].
• In both structures loss of Met ligation does not result in wholesale structural changes but is localized to a few residues adjacent to the now noncoordinating Met residue.
• Methionine heme-iron dissociation in ferricyts c is enhanced under alkaline conditions (pH ≥ 9.0) and is accompanied by a large structural rearrangement of the ligand loop [25], resulting in a Lys residue coordinating to the heme-iron [26].
• For P. versutus ferri-cyt c-550 NMR evidence is consistent with the presence of a single alkaline species with a de-protonated amino group of a side-chain Lys replacing the native M100 [3].
• This contrasts with the mitochondrial cyts c where a heterogenous mixture of alkaline species exists, reported to have a different coordinating Lys [26,27].
• Unfolding studies on ferricyts c reveal that dissociation of the Met heme-iron bond is the first step on the unfolding pathway.
• At pH values ≥7.0 certain ligand exchange events occur during unfolding which are akin to those for the ‘alkaline transition’.
• Moreover, it has been proposed that the structural units of cyts c which are responsible for the release of the Met ligand under alkaline conditions [28] are the same as those in the first step of the unfolding process [29-31].
• The spectroscopic properties of a cyte with a Lys residue replacing the native Met ligand were investigated with the M100K variant of P. versutus cyt c-550 [7].
• This mutation resulted in a mature protein which at neutral pH and in its ferric state exhibits similar spectroscopic properties to the single species observed at alkaline pH.
• This amongst other evidence suggested that K100 was coordinating the heme-iron [7].
• Interestingly, ligand exchange for the M100K variant at alkaline pH was not observed [7], suggesting that the ‘alkaline transition’ involving the dissociation of the axial ligand no longer occurs.
• The present study addresses the effect on structure and unfolding upon replacing the axial Met ligand with a Lys in the M100K variant of cyt c-550 from P. versutus.
• We describe three X-ray structures, one of the ferric wild type (wt) and two of the ferric M100K variant.
• The latter confirm Lys-heme coordination.
• Also the effect Lys-heme coordination has on the unfolding of the M100K variant is established by way of peroxidase activity assays and NMR spectroscopy.
• Results
• Structure determination and overall structure of the various ferricyt c-550 models
• For the M100K variant two X-ray datasets were collected.
• One at 295 K designated M100K room temperature (rt) to 1.95 Å resolution and one on a crystal at 100 K protected by a cryo-salt [32], and designated M100K cryo-cooled (cc) to 1.55 Å resolution.
• The structure was easily determined by molecular replacement using the structure of cyt c-550 from P. denitrificans (Protein Data Bank code: 1cot) strain LMD 22.21 [15,33].
• P. versutus cyt c-550 has a high homology with the cyt c-550 from P. denitrificans with only 22 amino acids differing of which 12 are conservative changes [4].
• The final models contained 121 amino acids, 32 (rt) and 138 (cc) water molecules and one heme group.
• An X-ray dataset was collected for the wt protein from a single crystal at room temperature to 1.90 Å resolution.
• Structure determination by molecular replacement was straightforward by using the M100K(rt) structure as a search model.
• Crystals of the wt protein contain one monomer in the asymmetric unit with the final wt model consisting of 119 amino acids, 50 water molecules and one heme group.
• In all models no density was observed for the N-terminal Gln.
• For the wt and M100K models density up to P120 and A122, respectively, was observed.
• No electron-density for the 13 residues after the C-terminal helix (helix 5, residues 108-117) starting at A122 was present.
• This part has been reported to be highly dynamic in solution [10].
• The statistics for data collection and refinement are summarized in Table 1.
• The program PROCHECK [34] was used to analyse conformational variations from the defined norms, with the quality of the Ramachandran plots [35] reported in Table 1.
• Electron density for the majority of side-chain atoms was clearly visible, although a number of surface exposed Lys residues have poor density and high thermal factors at the end of their side chain.
• The polypeptide fold of ferricyt c-550 observed in the crystal is composed of five a-helices (residues 5–14, 57-65, 73-81, 83-90 and 108-117) and two short stretches of β-strand (21-23 and 28-30), very similar to what was reported for the ferrous form in the solution state [10].
• A number of turns, consisting of two type I and four type II β-turns enable the polypeptide to wrap around the heme, Fig. 1.
• The average temperature factor fluctuations for the backbone atoms of the wt and M100K models are presented in Fig. 2.
• In all models, five regions exhibit above-average temperature fluctuations.
• In the wt protein these correspond to residues 23-35, 49-54, 64-65, 89-93 and 103-106 and are coloured blue in Fig. 1.
• For the M100K(rt) model a similar pattern of B-factors is observed although values are elevated compared to the wt and M100K (cc) model.
• This is most likely due to a less ordered crystal, which is also reflected in a slightly lower resolution of the data.
• After comparing the B-value patterns of the M100K(rt) and the wt-model, a noticeable difference can be observed for residues 90-100 (Fig. 2).
• This suggests enhanced flexibility in the ligand-loop region upon replacing M100 with a Lys residue.
• For the wt structure the regions exhibiting elevated B-values show good agreement with the dynamic data obtained in solution for both oxidation states of the protein [10,11].
• The heme environment of wt ferricyt c-550 and the M100K variant
• As in all cyts c the CXXCH heme binding motif is present.
• In P. versutus cyt c-550 the side-chain thiol groups of C15 and C18 form thioether linkages with the two vinyl groups of the heme.
• The N-terminal α-helix (helix 1) is distorted so the thioether bond from C15 to the heme can form (Fig. 1).
• Such a distortion appears unique to bacterial cyts c2 as it is not observed in the mitochondrial proteins where an additional amino acid residue in the helix is present [36].
• The Nε2 atom of the H19 side chain provides the fifth ligand to the heme-iron with a distance to the heme-iron of 2.01 Å and a H19_Nε2-iron-S M100 angle of 175°.
• H19 is further stabilized by a H-bond between the Nδ1 atom and the carbonyl oxygen of P37.
• In the wt structure the Sδ atom of M100 occupies the sixth coordination position to the heme-iron with a distance of 2.4 Å.
• This Sδ atom is also H-bonded to the hydroxyl group of Y79.
• This feature has been suggested to play a role in modulating the reduction potential.
• The length of this H-bond for P. versutus cyt c-550 is 3.8 Å.
• For cyt c2 from Rhodobacter capsulatus a similar distance is observed, yet the reduction potential is higher by 100 mV.
• In cyt c₂ of Rhodophila globiformis this distance is 3.2 Å, yet the potential is almost 200 mV higher.
• Thus it would appear that a clear correlation regarding the strength of this H-bonding interaction and the modulation of the reduction potential cannot easily be made.
• The heme group in the wt and M100K structures deviates from planarity and can be described as saddle shaped.
• Such a feature appears common to all cyts c, regardless of the nature of the sixth ligand to the heme-iron.
• In both the M100K structures clear electron density to the heme-iron is seen for the side chain of K100, confirming spectroscopic evidence that K100 is acting as a ligand to the heme-iron, Fig. 3.
• The bond lengths for the coordinating N³ and Nε atoms of K100 and H19 are 1.92 and 1.96 Å, respectively.
• For both coordinating atoms, the distance to the heme-iron is shorter than in the wt structure along with a decreased H19 Nε-iron-NK100 angle of 171°.
• Despite the shorter axial ligand distances the iron-pyrole nitrogen angles (175°) in the porphyrin ring remain approximately the same as in the wt structure (177°).
• The heme environment is further characterized by an extensive network of H-bonds involving buried water molecules, the heme propionate groups and side- and main-chain atoms of nearby residues (Fig. 4, Table 2).
• A noticeable difference in the heme cavity between the wt and both M100K structures is the presence in the latter of an additional water molecule adjacent to K100 (Fig. 4B).
• This water, wat6, is within H-bonding distance to the coordinating K100 N atom and also to wat3 (Table 2).
• Wat3 makes further H-bonding interactions with the carbonyl groups of V80 and F102 as noted also in the wt structure.
• The presence of wat6 therefore results in the formation of a buried two-water chain connecting the K100 ligand to two different backbone substructures of the protein (helix 3, V80 and the ligand loop, F102; Fig. 4B).
• Structural differences between wt ferricyt c-550 and the M100K(rt) variant
• The overall polypeptide fold of the wt protein is maintained for the M100K variant, with an r.m.s.d. for the backbone atoms of 0.4 Å.
• Significant deviations for main- and side-chain atoms in the ligand loop containing the K100 are however, observed.
• To accommodate K100 as a ligand a number of main-chain atoms are displaced relative to their positions in the wt structure.
• Although backbone deviations are detected at the start of the ligand loop, the largest changes are observed in the region between the amide nitrogen of K100 and the amide nitrogen of L104 (Fig. 4C).
• This movement results in a positional change of a surface water molecule (wat12).
• Wat12, found also in the wt structure, makes H-bonds to the backbone amides of T101 and F102.
• In the M100K structure wat12 moves 1.24 Å relative to its position in the wt structure and therefore maintains its H-bonding interactions with this region of the ligand loop (Fig. 4C).
• For a number of Lys residues complete electron density for the side chain was not observed.
• Nevertheless, for K97 density is observed up to the Cγ atom in wt and both M100K structures, allowing for good positional visualization of the side chain.
• From this density it is clear that the side-chain orientation of K97 in the two structures is different.
• In the wt structure the side chain slithers along the side of the protein surface, whereas in the M100K structure it points out into the solvent (Fig. 4C).
• The position of the K99 side chain is also different.
• In the wt structure a H-bonding interaction is made with the carbonyl oxygen of K54 (2.8 Å).
• Final refinement of the M100K(rt) structure positions the K99 side chain in such a way so as to increase this H-bonding distance to 3.1 Å. The different orientation of the