The sequence homologues of MT1 are found only in archaea and eukaryotes () and show no significant sequence similarity with other proteins of known structure. The MT1 monomer consists of a large dimerization domain (MT1-DD) and a small β-barrel auxiliary domain (MT1-CSD). Two monomers dimerize via the helical face of their MT1-DDs, burying 3740 Å2 (28%) of accessible surface area from each monomer (). The MT1-CSD is inserted in the second (αβ) loop region of the dimerization domain, and the two are connected by an α-helix and a 310 helix (). The β-barrel and a TIM barrel dimerization domains create a continuous 95 Å long positively charged surface for possible interaction with nucleic acid. Several strictly conserved residues are found on the dimerization as well as on MT1-DD/CSD interfaces.
Fig. 1 Sequence alignment of MT1 with proteins from a representative set of organisms using the program CLUSTALW.31 Completely conserved residues are highlighted in red; other conserved residues are highlighted in blue. Secondary structural elements are based (more ...)
Fig. 2 Overall structure of MT1 dimer. Stereoview is along non-crystallographic two-fold axis. Each monomer is separately colored and the knot region is marked in both monomers. The loop is dark blue and the C-terminal sequence threaded through the loop is red. (more ...)
The MT1-DD consists of 197 amino acids (residues 1–92 and 160–264) (). Analysis of structural homologs using the program DALI19
showed that the MT1-DD shares remarkable structural homology with TIM barrel enzymes, including E. coli
methylenetetrahydrofolate reductase (MTHFR) (PDB acc. no. 1B5T, Z score: 5.7, RMSD: 3.1 Å over 119 equivalenced residues), M. kandleri
coenzyme F420-dependent tetrahydromethanopterin reductase (PDB acc. no. 1EZW, Z score: 5.4, RMSD: 3.8 Å over 124 equivalenced residues), and rabbit muscle pyruvate kinase (PDB acc. no. 1A49, Z score: 4.6, RMSD: 3.4 Å over 117 equivalenced residues). Despite missing half of the structural elements of a TIM barrel, the MT1-DD five strands and four helices superpose with (α/β)8
arrangement of the MTHFR TIM barrel with an RMSD of 3.1 Å (β-strands 1, 2, 3, 7, and 8 and α-helices 1,2, 7, and 8 are present in MT1-DD, using the numbering convention from MTHFR . Helix 3's from MT1-DD and MTHFR do not overlap structurally because the MT1-DD α-helix is dragged out of position to connect with the β-strand 7 (). Hence, the 4th, 5th, and 6th (βα) units of MTHFR are missing in MT1-DD. Nevertheless, the MT1 dimer does not reconstitute complete TIM barrel structure. The existence of the MT1-DD stable partial TIM barrel strongly supports the notion that the ancestral TIM barrel was a half-barrel20
and suggests that the MT1-DD may be an early prototype of a TIM barrel. The β2 strand from the MT1 homolog from S. pombe
is flanked by a glycine and proline with a spacing and internal β-strand sequence consistent with other TIM barrels21
(). The MT1 structure appears also like a classical nucleotide binding Rossmann fold.22
However, MT1-DD has no conserved nucleotide binding motifs.
Fig. 3 Secondary structure and topology map of MT1 showing connectivity β-strands and α-helices. All secondary structural elements that are part of the TIM barrel are numbered according to the corresponding elements of MTHFR (see text). The remaining (more ...)
Fig. 4 Cα trace and stereo view of MT1-DD and MT1-CSD structural overlap. (a) The MT1-DD (red) is shown overlapped with the corresponding domain of MTHFR (green). Corresponding α-helices and β-strands are labeled as α and β, (more ...)
At its C-terminus, the dimerization domain contains a knot. The 35 C-terminal residues are threaded through a loop on a surface that connects α-helix 7 with β-strand 7 (, , and ). This is the first time that this architecture has been observed in a barrel-like structure. The knot region comprises a loop with a short β-strand that connects β-strand 8 with C-terminal α-helix 8. The crossover involves residues Val233, Asn234, Ala193, and Ser194 ( and ). The sequence that is threaded through the loop is not conserved with the exception of Asp230 and Pro237 on the C-side of the knot. There is also conserved proline residue within the loop (Pro195) that is located ~5 Å from the Pro237. Several hydrophobic residues are scattered throughout the sequence, and the length of the region varies among homologs (24–37 residues). The knot conformation is stabilized on N-site by Trp232, which appears to act as an anchor and on C-site by H-bond between Arg191 and Glu239. This region of MT1-DD is involved in dimer formation. Threading 35 residues through the loop region requires a major structural rearrangement (or cleavage and religation) of the protein main-chain.
Fig. 5 The MT1 trefoil knot. Residues 1–190 and 199–229 are shown in solvent accessible surface representation (1.4 Å radius). The knot loop (residues 191–198) is in blue and the polypeptide chain threaded through the loop (residues (more ...)
Only a few other proteins have a knotted fold.7–9
It is interesting that a very similar knot structure has been reported recently for the RrmA protein catalytic domain from Thermus thermophilus
, which is predicted to be a 2′-O-ribose methyltransferase.8
RrmA shares strong structural homology with MT1-DD including the knot region (PDB acc. no. 1IPA, Z score: 12.7, RMSD: 2.4 Å over 129 equivalenced residues). These proteins also show strikingly similar design. In MT1, a half-TIM barrel is fused with a putative cold-shock RNA-binding domain. In RrmA, the three-layer sandwich is fused with a eukaryotic ribosomal protein L30.8
Despite the structural and perhaps functional similarities, MT1 and RrmA share virtually no sequence similarity, and the proposed catalytic residues in RrmA are not conserved in MT1 family. Present data suggest that the machinery responsible for creating the knot structure is present in bacteria, archaea, and eukaryotes.
The charge properties of the surface of the MT1-DD, with a polar interior and a hydrophobic exterior, are unlike those found in TIM barrels. Within the barrel, MT1-DD has an atypical abundance of charged and polar residues that point into the center of the barrel. The polar nature is further strengthened by the third α3 helix and the loop linking strands β8 and β9, which shield several hydrophobic residues (data not shown). In contrast, its α-helical face is unusually hydrophobic and drives dimerization.
MT1 has an auxiliary domain inserted into a loop of the TIM barrel [ and ]. The auxiliary domain is 67 amino acids long and contains residues 93–159 in the MT1 protein [ and ]. The program DALI shows the this domain shares significant 3D structural similarity with three bacterial proteins: E. coli
major cold-shock protein CspA (PDB acc. no. 1MJC, Z score 5.6; RMSD: 2.5 Å over 58 equivalenced residues), E. coli
polyribonucleotide nucleotidyl transferase-S1 RNA-binding domain (PDB acc. no. 1SRO, Z score: 5.0; RMSD 2.3 Å over 52 equivalenced residues), Thermus thermophilus
S17 protein (PDB acc. no. 1FJF_Q, Z score 5.1, RMSD: 2.6 Å over 54 equivalenced residues). All four proteins are five-stranded antiparallel β-barrels that share the identical topology, known as the cold-shock domain23
. This type of structure is also classified as an oligonucleotide-binding (OB) fold. Preliminary data suggest that MT1 is not a cold-shock protein (Giometti and Tollakesen, personal communication). The electrostatic potential surface map drawn by using GRASP24
also indicates a potential nucleic acid-binding role. MT1 has a distinct positively charged face (data not shown). Although the C-terminal β-sheet residues of the barrels contribute a small portion of the positive charge, most of the charge is located in the MT1-CSDs. This charge distribution is similar to other nucleic acid-binding proteins with this fold.
It is likely that the MT1 binds single- or double-stranded RNA for the following reasons. First, MT1-CSD has significant structural similarity with CspA, which is an RNA chaperone that binds RNA to prevent hairpin formation for transcription antitermination,25,26
and with the ribosomal protein S17, which binds double-stranded regions of the 16s rRNA. Second, the gene for MT1 is located within a cluster of genes related to ribosomal function. However, we cannot rule out the possibility that MT1 binds DNA because the OB fold is found in many ssDNA-binding proteins,27
including the product of BRCA2 oncogene that contains three such units and binds single-stranded DNA.28
The MT1 structure, particularly the inserted domain, provides additional support to the observation that organisms accomplish complex tasks using modular protein design from a limited number of modules. There are at least two other examples of TIM barrel proteins that contain a similar insertion. Rabbit muscle pyruvate kinase contains a 9-stranded barrel inserted after the third βα loop. In that protein, the β-barrel domain forms part of the active site with another structurally unrelated domain.29
The Bacillus cereus
β-amylase TIM barrel contains a seven-stranded barrel, which forms the maltose-binding site and inserted at the C-terminus of the protein.30
In both instances, the inserted β-barrel domains are important for enzymatic catalysis.