Barley lipid-transfer protein complexed with palmitoyl CoA: the structure reveals a hydrophobic binding site that can expand to fit both large and small lipid-like ligands.

BACKGROUND: . Plant nonspecific lipid-transfer proteins (nsLTPs) bind a variety of very different lipids in vitro, including phospholipids, glycolipids, fatty acids and acyl coenzyme As. In this study we have determined the structure of a nsLTP complexed with palmitoyl coenzyme A (PCoA) in order to further our understanding of the structural mechanism of the broad specificity of these proteins and its relation to the function of nsLTPs in vivo. RESULTS: . 1H and 13C nuclear magnetic resonance spectroscopy (NMR) have been used to study the complex between a nsLTP isolated from barley seeds (bLTP) and the ligand PCoA. The resonances of 97% of the 1H atoms were assigned for the complexed bLTP and nearly all of the resonances were assigned in the bound PCoA ligand. The palmitoyl chain of the ligand was uniformly 13C-labelled allowing the two ends of the hydrocarbon chain to be assigned. The comparison of a subset of 20 calculated structures to an average structure showed root mean square deviations of 1.89 +/- 0.19 for all C, N, O, P and S atoms of the entire complex and of 0.57 +/- 0.09 for the peptide backbone atoms of the four alpha helices of the complexed bLTP. The four-helix topology of the uncomplexed bLTP is maintained in the complexed form of the protein. The bLTP only binds the hydrophobic parts of PCoA with the rest of the ligand remaining exposed to the solvent. The palmitoyl chain moiety of the ligand is placed in the interior of the protein and bent in a U-shape. This part of the ligand is completely buried within a hydrophobic pocket of the protein. CONCLUSIONS: . A comparison of the structures of bLTP in the free and bound forms suggests that bLTP can accommodate long olefinic ligands by expansion of the hydrophobic binding site. This expansion is achieved by a bend of one helix, HA, and by conformational changes in both the C terminus and helix HC. This mode of binding is different from that seen in the structure of maize nsLTP in complex with palmitic acid, where binding of the ligand is not associated with structural changes.

Structure in solution of a four-helix lipid binding protein.

Because of the low solubility of lipids in water, intercellular and intracellular pathways of lipid transfer are necessary, e.g., for membrane formation. The mechanism by which lipids in vivo are transported from their site of biogenesis (endoplasmatic reticulum and the chloroplasts) to their place of action is unknown. Several small plant proteins with the ability to mediate transfer of radiolabeled phospholipids in vitro from liposomal donor membranes to mitochondrial and chloroplast acceptor membranes have been isolated, and a protein with this ability, the nonspecific lipid transfer protein (nsLTP) isolated from barley seeds (bLTP), has been studied here. The structure and the protein lipid interactions of lipid transfer proteins are relevant for the understanding of their function, and here we present the three-dimensional structure in solution of bLTP as determined by NMR spectroscopy. The 1H NMR spectrum of the 91-residue protein was assigned for more than 97% of the protein 1H atoms, and the structure was calculated on the basis of 813 distance restraints from 1H-1H nuclear Overhauser effects, four disulfide bond restraints, from dihedral angle restraints for 66 phi-angles, 61 chi 1 angles, and 2 chi 2 angles, and from 31 sets of hydrogen bond restraints. The solution structure of bLTP consists of four well-defined alpha-helices A-D (A, Cys 3-Gly 19; B, Gly 25-Ala 38; C, Arg 44-Gly 57; D, Leu 63-Cys 73), separated by three short loops that are less well defined and concluded by a well defined C-terminal peptide segment with no observable regular secondary structure. For the 17 structures that are used to represent the solution structure of bLTP, the RMS deviation to an average structure is 0.63 A +/- 0.04 A for backbone atoms and 0.93 A +/- 0.06 A for all heavy atoms. The secondary structure elements and their locations in the sequence resemble those of nsLTP from two other plant species, wheat and maize, whose structures were previously determined (Gincel E et al, 1995, Eur J Biochem 226:413-422; Shin DH et al, 1995, Structure 3:189-199). In bLTP, the residues analogous to those in maize nsLTP that constitute the palmitate binding site are forming a similar hydrophobic cavity and a potential acyl group binding site. Analysis of the solution structure of bLTP and bLTP in complex with a ligand might provide information on the conformational changes in the protein upon ligand binding and subsequently provide information on the mode of ligand uptake and release. In this work, we hope to establish a foundation for further work of determining the solution structure of bLTP in complex with palmitoyl coenzyme A, which is a suitable ligand, and subsequently to outline the mode of ligand binding.

Mutational replacements of the amino acid residues forming the hydrophobic S4 binding pocket of subtilisin 309 from Bacillus lentus.

The amino acid side chains of Ile107, Leu126, and Leu135 participate in the formation of the important hydrophobic S4 binding pocket of the subtilisin Savinase. Ile107 and Leu126, located on each side of the pocket, point toward each other, and Leu135 is situated at the bottom of the pocket. These amino acid residues have been substituted for other hydrophobic amino acid residues by site-directed mutagenesis, and the resulting enzymes have been characterized with respect to their P4 substrate preferences. The Leu126-->Ala or Phe substitutions reduce kcat/KM for the hydrolysis of all substrates to around 5% without altering the substrate preference. It is concluded that Leu126 is an essential structural part of the pocket which cannot be replaced without seriously affecting catalysis, consistent with the fact that Leu126 is conserved among all subtilisins. In contrast, the Ile107-->Gly, Ala, Val, Leu, or Phe and Leu135-->Ala, Val, or Phe substitutions strongly influence the P4 substrate preference, and some of the mutants exhibit large specificity changes for particular substrates when compared to wild-type Savinase. The results can be rationalized on the basis of Ile107 and Leu135 being responsible for steric repulsion of branched aliphatic and aromatic P4 side chains, respectively. Leu135 exclusively interacts with aromatic P4 side chains, and its replacement with less bulky amino acid residues alleviates steric repulsion such that the activity toward this type of substrates is enhanced. Conversely, the introduction of a more bulky amino acid residue at position 135 produces more steric repulsion and reduces the activity toward substrates with aromatic P4 side chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Significance of hydrophobic S4-P4 interactions in subtilisin 309 from Bacillus lentus.

The subtilisins have an extended substrate binding cleft comprising at least 8 subsites. Two pockets at the S1 and S4 sites are particularly conspicuous, and the interactions between substrate and these two pockets are very important for the substrate specificity. Phe residues have mutationally been introduced at one of positions 102, 128, 130, and 132 of the subtilisin Savinase from Bacillus lentus to investigate the effects of introducing bulky groups along the rim of the S4 binding pocket. It is shown that the marked P4 preference of wild-type Savinase for aromatic groups is eliminated by the Gly102-->Phe and Ser128-->Phe mutations, indicating that bulky groups at positions 102 and 128 block the S4 binding site. In contrast, the activity toward hydrophilic P4 residues is not nearly as affected by these mutations, suggesting that the binding mode of the P4 side chain is dependent on its properties. Introduction of a bulky -CH2-S-CH2-CH2-pyridyl group at position 128, by mutational incorporation of Cys followed by chemical modification with 2-vinylpyridine, has essentially the same effect. The Ser130-->Phe mutation hardly affects the activity of the enzyme while the Ser-->Phe mutation at position 132 renders the preference for hydrophobic groups in P4 even more pronounced. This mutation furthermore affects the size of the S4 pocket. An analysis of double mutants at positions 132 and 104 suggests that the S4 region is flexible and is adjusted upon binding of substrates.

Mutational replacements in subtilisin 309. Val104 has a modulating effect on the P4 substrate preference.

The previous notion that the amino acid side chain at position 104 of subtilisins is involved in the binding of the side chain at position P4 of the substrate has been investigated. The amino acid residue Val104 in subtilisin 309 has been replaced by Ala, Arg, Asp, Phe, Ser, Trp and Tyr by site-directed mutagenesis. It is shown that the P4 specificity of this enzyme is not determined solely by the amino acid residue occupying position 104, as the enzyme exhibits a marked preference for aromatic groups in P4, regardless of the nature of the position-104 residue. With hydrophilic amino acid residues at this position, no involvement is seen in binding of either hydrophobic or hydrophilic amino acid residues at position P4 of the substrates. The substrate with Asp in P4 is an exception, as the preference for this substrate is increased dramatically by introduction of an arginine residue at position 104 in the enzyme, presumably due to a substrate-induced conformational change. However, when position 104 is occupied by hydrophobic residues, it is highly involved in binding of hydrophobic amino acid residues, either by increasing the hydrophobicity of S4 or by determining the size of the pocket. The results suggest that the amino acid residue at position 104 is mobile such that it is positioned in the S4 binding site only when it can interact favourably with the substrate's side chain at position P4.

Introduction of a free cysteinyl residue at position 68 in the subtilisin Savinase, based on homology with proteinase K.

Two subfamilies of the subtilisins, distinguished by the presence or absence of a free cysteinyl residue near the essential histidyl residue of the catalytic triad, are known. In order to evaluate the significance of the presence of this -SH group a cysteinyl residue has been introduced by site-directed mutagenesis into the cysteine-free subtilisin-like enzyme from Bacillus lentus, i.e. Savinase. The free cysteine affects the enzyme activity only slightly but renders it sensitive to mercurials presumably due to an indirect effect. The results indicate that the -SH group is not involved in catalysis.

A highly active and oxidation-resistant subtilisin-like enzyme produced by a combination of site-directed mutagenesis and chemical modification.

The subtilisins are known to be susceptible to chemical oxidation due to the conversion of Met222 into the corresponding sulfoxide. A number of derivatives with resistance towards oxidation have previously been prepared by replacement of this group with the other 19 amino acid residues. Unfortunately, the activities of these enzymes were of the order of 1-10% of that obtained with the wild-type enzyme. In contrast, the oxidation-labile cysteine mutant exhibited much higher activity, suggesting that this is associated with the presence of a sulphur atom in the amino acid at position 222. It is shown here that it is possible to maintain a sulphur atom in the amino acid at position 222 without the enzyme becoming labile towards oxidation. A subtilisin from Bacillus lentus, subtilisin 309, in which Met222 was replaced with a cysteinyl residue by site-directed mutagenesis was modified with thioalkylating reagents. Treatment of such enzyme derivatives with H2O2 revealed that their stabilities towards oxidation had increased significantly compared to both wild-type and unmodified [Cys222]subtilisin. One of the chemically modified enzyme derivatives, [Me-S-Cys222]subtilisin, exhibited a kcat/Km value of 56% of that obtained with the wild-type enzyme when assayed against the substrate Suc-Ala-Ala-Pro-Phe-NH-Ph-NO2 (Suc, succinyl) and it exhibited 89% activity when tested in an assay with dimethyl casein as a substrate. The corresponding values obtained for unmodified [Cys222]subtilisin were lower, i.e. 39% for the dimethyl casein activity and 46% for the kcat/Km for the hydrolysis of Suc-Ala-Ala-Pro-Phe-NH-Ph-NO2. This demonstrates the feasibility of replacing the oxidation-labile methionyl residue group in a subtilisin enzyme with a group stable towards oxidation without substantially reducing the activity.

Inactivation of carboxypeptidase Y by mutational removal of the putative essential histidyl residue.

Carboxypeptidase Y is a serine carboxypeptidase assumed to contain a catalytic triad similar to the serine endopeptidases. On the basis of the homology between various serine carboxypeptidases His-397 is suspected to be part of the catalytic triad. To test this it was exchanged with Ala and Arg by site-directed mutagenesis of the cloned PRC1 gene. The catalytic efficiency of the mutant enzymes were reduced by a factor of 2 X 10(4) and 7 X 10(2), respectively, confirming the key role of His-397 in catalysis. Treatment of Ala-397-CPD-Y with Hg++ or CNBr, hence modifying Cys-341 located in the vicinity of the active site abolished the residual activity of the enzyme, indicating an additional involvement of this residue in catalysis.

Chemical modifications of a cysteinyl residue introduced in the binding site of carboxypeptidase Y by site-directed mutagenesis.

It is demonstrated that site-directed mutagenesis successfully can be combined with chemical modification creating enzyme derivatives with altered properties. A methionyl residue located in the S1' binding site of carboxypeptidase Y was replaced by a cysteinyl residue and the mutant enzyme was isolated and modified with various alkylating and thioalkylating reagents. Treatment of the mutant carboxypeptidase Y with bulky reagents like phenacyl bromide and benzyl methanethiolsulfonate caused a drastic reduction in the activity towards substrates with bulky leaving groups in the P1' position, i.e. -OBzl, -Val-NH2 and amino acids (except -Gly-OH), while substrates with small groups in that position, i.e. -OMe and -NH2, were hydrolysed with increased rates. The presence of a positive charge, in addition to a bulky group, had a further adverse effect on the activity towards substrates with large leaving groups, whereas the activity towards those with small leaving groups remained unaffected by such a group. The derivatives obtained by modification of the mutant enzyme with benzyl methanethiolsulfonate and methyl methanethiolsulfonate were effective in deamidations of peptide amides and peptide synthesis reactions, respectively.