On the Relevance of the Met-turn Methionine in Metzincins*

Evolutionary conservation of structural elements in proteins usually results from stringent steric requirements for function. Specific structures enable particular chemical groups to be placed appropriately in three-dimensional space for reaction, transport, regulation, and scaffolding (). One such case of conserved structural elements is the Met-turn, found in the catalytic domains of all structurally characterized families of the metzincin clan of zinc-dependent metallopeptidases. These include astacins, ADAMs/adamalysins, serralysins, matrix metalloproteinases (MMPs), leishmanolysins, snapalysins, and pappalysins (, , , ). The catalytic domains span between ∼130 and ∼260 residues and fold into globular moieties, which are divided by an active site cleft into an upper N-terminal and a lower C-terminal subdomain when viewed in the standard orientation (Fig. 1A) (). The N-terminal subdomain comprises a β-sheet and two helices, the backing helix and the active-site helix, as the minimum common core of repetitive secondary structure elements (). The latter helix includes the first stretch of a zinc-binding consensus sequence, HEXXHXXG/NXXH/D (amino acid one-letter code), which includes three protein ligands of the catalytic zinc and the general base/acid glutamate for catalysis. Features of the C-terminal subdomain common to all metzincins include a C-terminal α-helix and the Met-turn (Fig. 1B). In the course of evolution, distinct structural elements have been introduced into this consensus minimal scaffold for each family, such as helices, strands, structural zinc- and calcium-binding sites, and additional domains (, , , ). For example, in the smallest metzincin representative reported for its structure, 132-residue Streptomyces caespitosus snapalysin, only one extra calcium-binding site and a short helix in the N-terminal subdomain are found (Fig. 1A). The result of such insertions is that, overall, the families have evolved along separate pathways. This is reflected by sequence identities well below twilight values to discriminate between similar and dissimilar structures (20–35%) (), and most sequence alignment protocols only identify the extended zinc-binding sequence and flanking sequence stretches (). However, the existence of the aforementioned common structural core elements confirms that all of these families are homologous, thus underlining that structural relatedness is more conserved than sequence identity ().

The Met-turn is a conserved 1,4-β-turn of type I that contains a methionine at position 3, which is separated from the third zinc-binding histidine/aspartate by connecting segments of 6–53 amino acids in the different metzincin structures (, ). This means that the chain traces of the distinct prototypes strongly diverge after the third zinc ligand, but they all converge at the Met-turn. In all structures analyzed, this type I turn is superimposable in space, with the same conformation of the methionine side chain, with dihedral angles χ₁, χ₂, and χ₃ adopting values in the ranges 282–309°, 276–317°, and 286–328°, respectively (Fig. 1B). This residue lies underneath the catalytic zinc. However, the distance from the side-chain atoms to the metal is far too great, and the orientation of the lone electron pairs of the methionine sulfur is incompatible with a metal interaction. This strict conservation of a residue imbedded in a small structural element spanning four residues would suggest a pivotal role in structural and functional integrity because methionine is not a residue particularly conserved during evolution (). However, mutation studies performed on several metzincin families revealed disparate results.

Replacement of the Met-turn methionine by selenomethionine in matrix metalloproteinase 8 (MMP-8) gave rise to a 50% decrease in catalytic efficiency and diminished conformational stability, but did not affect the crystal structure (). Similar studies on MMP-2 showed unaltered activity for the serine and leucine mutants but complete loss of activity and enhanced susceptibility to proteolytic degradation for the cysteine mutant (). In serralysins, substitution of difluoromethionine for methionine revealed no significant differences in activity or in the result of differential scanning calorimetry in Pseudomonas aeruginosa alkaline protease (). In contrast, studies on Erwinia chrysanthemi PrtC revealed lower levels of protein expression and diminished catalytic efficiency toward resorufin-casein for the leucine (85% of the wild type), isoleucine (50%), and alanine (23%) mutants and subtle changes in the crystal structure at the active site of the first two mutants and a cysteine mutant. However, major structural rearrangement and destabilization of the zinc site and an adjacent protein segment spanning ∼80 residues were observed in the alanine mutant. In the histidine mutant crystal structure, an alternative zinc-binding site was found below the functional site (, ). With respect to ADAMs/adamalysins, mutation of methionine in tissue necrosis factor α-converting enzyme to isoleucine, leucine, or serine impaired the ectodomain shedding of physiological substrates (). In the case of the astacin family, an alanine mutant of crayfish astacin showed significantly lower activity in gelatin zymography but similar thermal stability in CD spectroscopy. Finally, a leucine mutant of pregnancy-associated plasma protein A (PAPP-A) from the pappalysin family rendered expression levels similar to wild type, but only ∼5% of its capacity to cleave a physiological protein substrate ().

To examine further the role of the Met-turn methionine in metzincins, we studied a distinct representative, Methanosarcina acetivoras ulilysin, and mutated its Met²⁹⁰ to 10 different residues. We analyzed recombinant expression levels of these proulilysin variants, (autolytic) activation through limited proteolysis, and activity of the mature ulilysin forms. We further performed thermal shift assays of zymogens and mature enzymes to study structural stability, and we managed to crystallize and solve the structure of the mature forms of two of these mutants, ulilysin M290L and M290C.

The strict conservation of the Met-turn and its methionine among metzincin structures has intrigued structural biologists since its discovery, and it contributed to the name of this metallopeptidase clan (). Several methionine-replacement studies had shown that this residue is important for fold and function. Our results on 10 ulilysin mutants support its functional relevance. All mutants were expressed and purified, although in varying amounts, thus suggesting at least partial defects in synthesis, secretion, and stability. No mutant proved more stable or active than the wild type. We found an inverse correlation between function/stability and deviation from the optimal size as granted by methionine. In addition, side-chain hydrophobicity was also found to be indispensable because all hydrophilic mutants assayed displayed very low resistance to thermal denaturation. In most cases, they could not be activated but were degraded instead. The next-to-best residues were selenomethionine and leucine, which occupied approximately the same space in the three-dimensional structure as methionine and were similarly hydrophobic and functional. However, the latter was only functional as the wild type under the assumption of an unusual rotamer, thus indicating a suboptimal qualification. The next best option was M290C, which showed lower activity but shared hydrophobicity with the former residues and in which the smaller side chain was offset by the recruitment of a buried solvent molecule. Further ranks were occupied by phenylalanine and valine, with deviating side-chain sizes but similar hydrophobicity. We concluded that there is little margin for variation from a fully competent structure in ulilysin, and activity comparable with the wild type was only observed in structurally equivalent molecules. This was further underlined by detailed inspection of the zinc site in metzincin structures, which revealed a compact core made up by six conserved hydrogen bonds plus a hydrophobic interaction made up by the residues of the zinc-binding consensus sequence and the two central residues of the Met-turn. The latter is a plug that must be snugly inserted into the zinc site to render it functional, and this pinpoints methionine as the “best adapted” residue, which must present a particular combination of conserved side-chain dihedral angles. Mutations entailing significant changes in the size and chemical nature of the side chain would prejudice the functionality of the Met-turn plug, thus giving rise to molecules that were not (auto)activatable, nor crystallizable, or only residually active in best cases. Accordingly, our studies provide an explanation for the absolute conservation of the methionine during evolution, based on steric and chemical requirements for structure and function. Finally, although these results are consistent with most reports on other metzincins, it cannot be ruled out that slight differences in selected structural elements may be observed in certain families, in particular if they do not significantly affect the central zinc-binding core structure.