Helix movements and the reconstruction of the haem pocket during the evolution of the cytochrome c family

Analysis of cytochromes c (tuna), c2 (Rhodospirillum rubrum), c550 (Paracoccus denitrificans) and c551 (Pseudomonas aeruginosa) shows that they contain 48 residues identifiable as homologous from superposition of the structures. The other 34 to 64 residues are in loops that vary greatly in sequence, length and conformation, or in alpha-helices that are found in only some of the structures. Of the 48 homologous residues, 17 are in three segments which pack onto the haem faces. In all four structures, these segments have the same conformations, and the same locations relative to the haem. The other 31 residues are in three alpha-helices which are in contact with each other. These form the back and one side of the haem pocket. In cytochrome c551 the positions of the three alpha-helices have shifted and rotated, in comparison with cytochromes c and c2, by up to 5 A and 25 degrees relative to the haem. These shifts, facilitated by mutations at the helix-helix interfaces, are related to the reconstruction of the propionic acid side of the haem pocket described by Almassy & Dickerson (1978). Together these effects produce alternative structures for the haem pocket. This mechanism of adaptation to mutation contrasts with that observed in the globins. In the globins, mutations also produce changes in helix interfaces and shifts of packed helices, but in the globins these shifts are coupled to conserve the structure of the haem pocket.     

Determinants of a protein fold. Unique features of the globin amino acid sequences

The three-dimensional structures of globins are known, from crystallographic analyses, to be very similar. Their amino acid sequences, however, differ greatly. Only two residues are absolutely conserved in all sequences, and the residue identities of some pairs of sequences are only 16%. We have determined the nature and exact extent of the sequence variations and the extent to which the conserved features of the globin sequences are unique to this family. The 226 globin sequences now known were aligned and analysed. Because distantly related protein sequences cannot be aligned correctly without the use of structural data, we developed a method that incorporated structural information into the alignment procedure. Analysis of the aligned sequences show that: (1) Although individual chains vary in size between 132 and 157 residues, deletions and insertions result in there being only 102 residue sites common to all globins. These sites form six separate regions. Insertions and deletions between these regions means that their separations can vary in different sequences. (2) Within the conserved regions there are 32 sites that almost always contain hydrophobic residues. In the known structures, these sites are in the protein interior. We measured the variations in the size of the residues that occur in the 226 sequences at these sites. At six sites the residues differ in size by less than 40 A3, at 11 sites they differ by 40 to 100 A3, and at 15 sites they differ by more than 100 A3. There are two other conserved buried sites: one contains the His linked to the haem iron and the other usually contains a His involved with the haem ligand. (3) Within the conserved regions there are another 32 sites that are almost always occupied by charged, polar or small non-polar (Gly or Ala) residues. In the known structures, these sites are on the protein surface. To determine the extent to which the conserved features found for the globin sequences are unique to that protein family, the following procedure was used. The six conserved regions, and the residue restrictions that occur at the 66 sites within these regions, were encoded into two "templates". One was based only on the sequences so far determined; the other was extended to include as yet unobserved substitutions that seemed plausible on the basis of size, hydrophobicity and polarity. Each of the 3286 non-globin sequences in the data bank was then examined by a computer program to see how closely it could be matched to these templates.(ABSTRACT TRUNCATED AT 400 WORDS)     

NH2-terminal sequence analyses of four rat hepatic microsomal cytochromes P-450

Cytochromes P-450f, P-450g, P-450h, and P-450i are four hepatic microsomal hemoproteins that have been purified from adult rats. Whereas cytochromes P-450g and P-450h appear to be male-specific hemoproteins, cytochrome P-450i is apparently a female-specific enzyme purified from untreated adult female rats. Cytochrome P-450f has been purified from adult male and female rats with equivalent recoveries. Amino-terminal sequence analyses of the first 15-20 amino acid residues of each of these cytochromes P-450 has been accomplished in the current investigation. Each protein possesses a hydrophobic leader sequence consisting of 65-87% hydrophobic amino acids, and only one charged amino acid (Asp) in the amino-terminal region. Although differences in the amino-terminal sequences of cytochromes P-450f, P-450g, P-450h, and P-450i are identified, these hemoproteins all begin with Met-Asp, and marked structural homology is observed among certain of these enzymes. Cytochromes P-450g and P-450h, two male-specific proteins, have 11-12/15 identical residues with cytochrome P-450i, a female-specific isozyme. Cytochromes P-450f and P-450h have 16/20 identical amino-terminal residues. Only limited sequence homology is observed between the amino-terminal sequences of cytochromes P-450f-i compared to rat liver cytochromes P-450a-e. The results demonstrate that cytochromes P-450f, P-450g, P-450h, and P-450i are isozymic to each other and five additional rat hepatic microsomal cytochrome P-450 isozymes (P-450a-e).     

A common mechanism for ATP hydrolysis in ABC transporter and helicase superfamilies

ABC (ATP-binding cassette) transporters and helicases are large superfamilies of seemingly unrelated proteins, whose functions depend on the energy provided by ATP hydrolysis. Comparison of the 3D structures of their nucleotide-binding domains reveals that, besides two well-characterized ATP-binding signatures, the folds of their nucleotide-binding sites are similar. Furthermore, there are striking similarities in the positioning of residues thought to be important for ATP binding or hydrolysis. Interestingly, structures have recently been obtained for two ABC proteins that are not involved in transport activities, but that have a function related to DNA modification. These ABC proteins, which contain a nucleotide-binding site akin to those of typical ABC transporters, might constitute the missing link between the two superfamilies.     

Non-functional conserved residues in globins and their possible role as a folding nucleus.

Structure-based sequence alignment of 728 sequences of different globin subfamilies shows that in each subfamily there are two clusters of consensually conserved residues. The first is the well-known "functional" cluster which includes six heme-binding conserved residues (Phe CD1, His F8; aliphatic E11, FG5; hydrophobic F4, G5) and seven other conserved residues (Pro C2; aliphatic H19; hydrophobic B10, B13, B14, CD4, E4) that do not bind the heme but belong to its immediate neighborhood. The second cluster revealed here (aliphatic A8, G16, G12; aromatic A12; hydrophobic H8 and possibly H12) is distant from the heme. It is entirely non-polar and includes one turn (i, i+4 positions) from each of helices A, G, and H. It is known that A, G, and H helices formed at the earliest stage of apomyoglobin folding remain relatively stable in the equilibrium molten globule state, and are likely to be tightly packed with each other in this state. We have shown the existence of two similar conserved clusters in c -type cytochromes, heme-binding and distal from the heme. The second cluster in c -cytochromes includes one turn from each of the N and C-terminal alpha-helices. These N and C-terminal helices in cytochrome c are formed at the earliest stage of protein folding, remain relatively stable in the molten globule state, and are tightly packed with each other in this state, similar to the observed behavior of the globins. At least these two large protein families (c -type cytochromes and globins) have a close similarity in the existence and mutual positions of non-functional conserved residues. We assume that non-functional conserved residues are requisite for the fast and correct folding of both of these protein families into their stable 3D structures.     

Evolutionarily conserved substrate substructures for automated annotation of enzyme superfamilies

The evolution of enzymes affects how well a species can adapt to new environmental conditions. During enzyme evolution, certain aspects of molecular function are conserved while other aspects can vary. Aspects of function that are more difficult to change or that need to be reused in multiple contexts are often conserved, while those that vary may indicate functions that are more easily changed or that are no longer required. In analogy to the study of conservation patterns in enzyme sequences and structures, we have examined the patterns of conservation and variation in enzyme function by analyzing graph isomorphisms among enzyme substrates of a large number of enzyme superfamilies. This systematic analysis of substrate substructures establishes the conservation patterns that typify individual superfamilies. Specifically, we determined the chemical substructures that are conserved among all known substrates of a superfamily and the substructures that are reacting in these substrates and then examined the relationship between the two. Across the 42 superfamilies that were analyzed, substantial variation was found in how much of the conserved substructure is reacting, suggesting that superfamilies may not be easily grouped into discrete and separable categories. Instead, our results suggest that many superfamilies may need to be treated individually for analyses of evolution, function prediction, and guiding enzyme engineering strategies. Annotating superfamilies with these conserved and reacting substructure patterns provides information that is orthogonal to information provided by studies of conservation in superfamily sequences and structures, thereby improving the precision with which we can predict the functions of enzymes of unknown function and direct studies in enzyme engineering. Because the method is automated, it is suitable for large-scale characterization and comparison of fundamental functional capabilities of both characterized and uncharacterized enzyme superfamilies.     

Identification of selectivity-determining residues in cytochrome P450 monooxygenases: a systematic analysis of the substrate recognition site 5

The large and diverse family of cytochrome P450 monooxygenases (CYPs) was systematically analyzed to identify selectivity- and specificity-determining residues in the substrate recognition site 5, which is located in close vicinity to the heme center. A positively charged heme-interacting residue was identified in the structures of 29 monooxygenases and in 97.7% of the 6379 CYP sequences investigated here. This heme-interacting residue restricts the conformation of the substrate recognition site 5 and is preferentially located at position 10 or 11 after the conserved ExxR motif (in 94.4% of the sequences), in 3.3% of the sequences at position 9 or 12. As a result, a classification by the position of the heme-interacting residue allows to predict residues that are closest to the heme center and restrict its accessibility. In 98.4% of all CYP sequences a preferentially hydrophobic residue is located at position 5 after the ExxR motif that is predicted to point close to the heme center. Replacing this residue by hydrophobic residues of different size has been shown to change substrate specificity and regioselectivity for CYPs of different superfamilies. Twenty-seven percent of all CYPs are predicted to contain a second selectivity-determining residue at position 9 after the ExxR motif that can be identified by the pattern EXXR-X(7)-{P}-x-P-[HKR].     

Revealing the set of mutually correlated positions for the protein families of immunoglobulin fold

In this study, I explain the observation that a rather limited number of residues (about 10) establishes the immunoglobulin fold for the sequences of about 100 residues. Immunoglobulin fold proteins (IgF) comprise SCOP protein superfamilies with rather different functions and with less than 10% sequence identity; their alignment can be accomplished only taking into account the 3D structure. Therefore, I believe that discovering the additional common features of the sequences is necessary to explain the existence of a common fold for these SCOP superfamilies. We propose a method for analysis of pair-wise interconnections between residues of the multiple sequence alignment which helps us to reveal the set of mutually correlated positions, inherent to almost every superfamily of this protein fold. Hence, the set of constant positions (comprising the hydrophobic common core) and the set of variable but mutually correlated ones can serve as a basis of having the common 3D structure for rather distinct protein sequences.     

Structure–Critical Distribution of Aromatic Residues in the Fibronectin Type III Protein Family

Over a thousand individual Fibronectin type III (FnIII) domain sequences, extracted from more than 60 different FnIII-dependent protein super-structures, were downloaded from curated database resources. Three regions of extreme sequence conservation within the well-characterized FnIII β-sandwich structure were respectively defined by near absolute conservation of a tryptophan (Trp) in β-strand-B, tyrosines (Tyr) in both β-strand-C and β-strand-F, and a leucine (Leu) residue in the unstructured region immediately preceding β-strand-F. Employing these four conserved landmarks, the entire FnIII sequence dataset was vertically registered to align the three conserved regions, and the cumulative distribution of all other amino acid functionality was determined and plotted relative to these landmark residues. Conserved aromatic sites were each found to be flanked by aliphatic residues that assure localization of these sites to the inaccessible hydrophobic interface between major sheet structures. Mapping the location of conserved aromatic sites in numerous PDB structures demonstrated the consistent pair-wise co-localization of the indole side-chain of the conserved strand-B Trp site to within 0.35 nm of the phenolic side-chain of the strand-C Tyr site located 8–14 amino acids distal. Likewise, the side-chain of the strand-F Tyr site co-localized to within 0.45 nm of the aliphatic side-chain of the conserved Leu that uniformly precedes it by six residues. While classic hydropathy-based theories would deem the “burying” of Tyr and Trp side-chains and/or their association with hydrophobic FnIII core residues thermodynamically unnecessary, alternative contributions of conserved Trp and Tyr residues, and particularly the role of the absolutely conserved tyrosine phenolic –OH in native FnIII structure–function are considered. A more global role for conserved FnIII aromaticity is also discussed in light of the aromatic conservation observed in other well-established protein families.     

A dedicated haem lyase is required for the maturation of a novel bacterial cytochrome c with unconventional covalent haem binding

In bacterial c-type cytochromes, the haem cofactor is covalently attached via two cysteine residues organized in a haem c-binding motif. Here, a novel octa-haem c protein, MccA, is described that contains only seven conventional haem c-binding motifs (CXXCH), in addition to several single cysteine residues and a conserved CH signature. Mass spectrometric analysis of purified MccA from Wolinella succinogenes suggests that two of the single cysteine residues are actually part of an unprecedented CX15CH sequence involved in haem c binding. Spectroscopic characterization of MccA identified an unusual high-potential haem c with a red-shifted absorption maximum, not unlike that of certain eukaryotic cytochromes c that exceptionally bind haem via only one thioether bridge. A haem lyase gene was found to be specifically required for the maturation of MccA in W. succinogenes. Equivalent haem lyase-encoding genes belonging to either the bacterial cytochrome c biogenesis system I or II are present in the vicinity of every known mccA gene suggesting a dedicated cytochrome c maturation pathway. The results necessitate reconsideration of computer-based prediction of putative haem c-binding motifs in bacterial proteomes.     

Conformational homologies among cytokines: Interleukins and colony stimulating factors

Some 30 cytokine amino acid sequences (mainly interleukins, colony stimulating factors and tumor necrosis factors) have been examined for evidence of secondary structure as well as longer-range interactions of a type likely to lead to stable α-helical bundles. Most, though not all, of the cytokines examined have a high predicted α-helical content (40–60%) and quasi-repeating heptads containing i/i+3 apolar periodicities. This major subset of the cytokines is predicted to be characterized by molecules in which 4-α-helical bundles with an average length of 25Å are the most marked conformational features. Based on these conclusions, we suggest structures for huG-CSF, huGM-CSF and muIL-5 in which defined loop segments at the ends of helical bundles are the most likely sites for binding and recognition by specific cell receptors. As such, they provide a means for testing or refining the three working models we have defined, using currently available methods of site-directed substitution and deletion mutagenesis, as well as synthetic peptides corresponding to the proposed loop sequences and the use of monoclonal antibodies of defined epitopic specificity. The structure arrived at for huGM-CSF is consistent with the limited data currently available concerning the residues which are important for binding and activity.     

A hydrophobic spine stabilizes a surface-exposed α-helix according to analysis of the solvent-accessible surface area

Background

Most of hydrophilic and hydrophobic residues are thought to be exposed and buried in proteins, respectively. In contrast to the majority of the existing studies on protein folding characteristics using protein structures, in this study, our aim was to design predictors for estimating relative solvent accessibility (RSA) of amino acid residues to discover protein folding characteristics from sequences.

Methods

The proposed 20 real-value RSA predictors were designed on the basis of the support vector regression method with a set of informative physicochemical properties (PCPs) obtained by means of an optimal feature selection algorithm. Then, molecular dynamics simulations were performed for validating the knowledge discovered by analysis of the selected PCPs.

Results

The RSA predictors had the mean absolute error of 14.11% and a correlation coefficient of 0.69, better than the existing predictors. The hydrophilic-residue predictors preferred PCPs of buried amino acid residues to PCPs of exposed ones as prediction features. A hydrophobic spine composed of exposed hydrophobic residues of an α-helix was discovered by analyzing the PCPs of RSA predictors corresponding to hydrophobic residues. For example, the results of a molecular dynamics simulation of wild-type sequences and their mutants showed that proteins 1MOF and 2WRP_H16I (Protein Data Bank IDs), which have a perfectly hydrophobic spine, have more stable structures than 1MOF_I54D and 2WRP do (which do not have a perfectly hydrophobic spine).

Conclusions

We identified informative PCPs to design high-performance RSA predictors and to analyze these PCPs for identification of novel protein folding characteristics. A hydrophobic spine in a protein can help to stabilize exposed α-helices.

     

The amino acid sequence of cytochromes c-551 from three species of Pseudomonas

The amino acid sequences of the cytochromes c-551 from three species of Pseudomonas have been determined. Each resembles the protein from Pseudomonas strain P6009 (now known to be Pseudomonas aeruginosa, not Pseudomonas fluorescens) in containing 82 amino acids in a single peptide chain, with a haem group covalently attached to cysteine residues 12 and 15. In all four sequences 43 residues are identical. Although by bacteriological criteria the organisms are closely related, the differences between pairs of sequences range from 22% to 39%. These values should be compared with the differences in the sequence of mitochondrial cytochrome c between mammals and amphibians (about 18%) or between mammals and insects (about 33%). Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 50015 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 5.     

Haem staining in gels, a useful tool in the study of bacterial c-type cytochromes

c-Type cytochromes are the only cytochromes to retain quantitatively their haem during SDS gel electrophoresis and can be identified in complex mixtures by their haem peroxidase activity. Although weak staining bands may be due to residual haem attachment to b-type cytochromes or to migration of haem, these effects could be abolished by prior extraction with organic solvent. The colour yield of haem staining allowed an estimate of the relative amounts of a particular cytochrome, particularly if loadings were below 50 pmol. At greater loadings, a plateau of colour development was observed. Freshly made gels gave much poorer colour development. The haem-staining method was shown to be useful in three particular areas of study in bacterial respiration. Firstly, it allows assessment of the results of sphaeroplast formation in gram negative bacteria. Secondly, quantitation of the haem stain was useful in the investigation of the induction effects of growth conditions on c-type cytochromes. Thirdly, the interpretation of complex chromatographic profiles was greatly simplified by the use of haem-stained SDS electrophoretic gels.     

The primary structure of cytochrome c from the rust fungus Ustilago sphaerogena

1. The complete amino acid sequence of cytochrome c from the basidiomycete Ustilago sphaerogena was determined from the amino acid compositions and sequences of either tryptic or chymotryptic peptides, and in homology with at least thirty other established sequences of cytochrome c. 2. The primary structure of the molecule bears all of the characteristics of a mammalian-type cytochrome c, showing the typical clustered distribution of hydrophobic and basic residues with a single polypeptide chain of 107 residues. 3. Like all other fungal cytochromes c, it possesses a free N-terminus, and one less residue at the C-terminus than vertebrate cytochromes c. The region of residues 70-80 is strictly conserved, as is histidine at position 18. Position 26 is occupied by an asparagine residue, in contrast to histidine which occurs at this location in most of the known sequences of mammalian-type cytochromes c. 4. In contrast to some other fungal and plant cytochromes c of known primary structures, the Ustilago cytochrome c molecule does not contain trimethyl-lysine. 5. The sequence of Ustilago cytochrome c differs from the sequences of human, horse, chicken, tuna, wheat, and baker's yeast proteins at loci 47, 43, 44, 44 and 38 respectively.     

Redox potentials of the photosynthetic bacterial cytochromes c2 and the structural bases for variability.

The cytochromes c2 of the Rhodospirillaceae show a much greater variation in redox potential and its pH dependence than the mitochondrial cytochromes c that have been studied. It is proposed that the range of redox potential for cytochromes c2 functioning as the immediate electron donor to photo-oxidised bacteriochlorophyll may be 345-395 mV at pH 5. Closely related cytochromes c2 with different redox potentials show patterns of amino acid substitution which are consistent with changes in hydrophobicity near the haem being at least a partial determinant of redox potential. More distantly related cytochromes are difficult to compare because of the large number of amino acid substitutions and the probability that there are subtle changes in overall peptide chain folding. The redox potential versus pH curves can be analysed in terms of either one ionisation in the oxidised form or two in the oxidised form and one in the reduced. The pK in the oxidised form at higher pH values can be correlated with the pK for the disappearance or shift of the near infrared absorption band located near 695 nm. The structural bases of these ionisations are not known but the possible involvement of the haem propionate residues is discussed.     

Redox potentials of the photosynthetic bacterial cytochromes c2 and the structural bases for variability

The cytochromes c₂ of the Rhodospirillaceae show a much greater variation in redox potential and its pH dependence than the mitochondrial cytochromes c that have been studied. It is proposed that the range of redox potential for cytochromes c₂ functioning as the immediate electron donor to photo-oxidised bacteriochlorophyll may be 345–395 mV at pH 5.Closely related cytochromes c₂ with different redox potentials show patterns of amino acid substitution which are consistent with changes in hydrophobicity near the haem being at least a partial determinant of redox potential. More distantly related cytochromes are difficult to compare because of the large number of amino acid substitutions and the probability that there are subtle changes in overall peptide chain folding.The redox potential versus pH curves can be analysed in terms of either one ionisation in the oxidised form or two in the oxidised form and one in the reduced. The pK in the oxidised form at higher pH values can be correlated with the pK for the disappearance or shift of the near infrared absorption band located near 695 nm. The structural bases of these ionisations are not known but the possible involvement of the haem propionate residues is discussed.     

How different amino acid sequences determine similar protein structures: The structure and evolutionary dynamics of the globins

To determine how different amino acid sequences form similar protein structures, and how proteins adapt to mutations that change the volume of residues buried in their close-packed interiors, we have analysed and compared the atomic structures of nine different globins. The homology of the sequences in the two most distantly related molecules is only 16%.The principal determinants of three-dimensional structure of these proteins are the approximately 59 residues involved in helix to helix and helix to haem packings. Half of these residues are buried within the molecules. The observed variations in the sequence keep the side-chains of buried residues non-polar, but do not maintain their size: the mean variation of the volume among homologous amino acids is 56 ų.Changes in the volumes of buried residues are accompanied by changes in the geometry of the helix packings. The relative positions and orientations of homologous pairs of helices in the globins differ by rigid body shifts of up to 7 Å and 30 °. In order to retain functional activity these shifts are coupled so that the geometry of the residues forming the haem pocket is very similar in all the globins.We discuss the implications of these results for the mechanism of protein evolution.     

Plant peroxidases. Their primary, secondary and tertiary structures, and relation to cytochrome c peroxidase

The amino acid sequences of the 51% different horseradish peroxidase HRP C and turnip peroxidase TP 7 have previously been completed by us, but the three-dimensional structures are unknown. Recently the amino acid sequence and the crystal structure of yeast cytochrome c peroxidase have appeared. The three known apoperoxidases consist of 300 +/- 8 amino acid residues. The sequences have now been aligned and show 18% and 16% identity only, between the yeast peroxidase and plant peroxidase HRP C and TP 7, respectively. We show that different structural tests all support similar protein folds in plant peroxidases and yeast peroxidase and, therefore, a common evolutionary origin. The following tests support this thesis: (a) predicted helices in the plant peroxidases follow the complex pattern observed in the crystal structure of cytochrome c peroxidase; (b) their hydropathic profiles are similar and agree with observed buried and exposed peptide chain in cytochrome c peroxidase; (c) half-cystines which are distant in the amino acid sequence of plant peroxidases become spatial neighbours when fitted into the cytochrome c peroxidase model; (d) the two-domain structure proposed from limited proteolysis of apoperoxidase HRP C is observed in the crystal structure of cytochrome c peroxidase. The similarities and differences of the plant and yeast peroxidases and the reactive side chains of a plant peroxidase active site are described. The characteristics of Ca2+-binding sequences, derived from several superfamilies, are applied to predict the Ca2+-binding sequences in plant peroxidases.