In the beginning, we need to choose the sequences for the alignment. To start, we write the name of the protein (BchD) into the UniProt
search window, and we will be taken to a list of sequences of the protein from different organisms
The site shows a large number of sequences. You may notice on the left, where it says “Filtered by,” that there are “Reviewed
” and “Unreviewed
” sequences. This is one of my favorite features - when there is a sufficient number of reviewed sequences, I usually choose them for further analysis. These sequences have been verified to be what we expect them to be. There are many automatically annotated sequences among the Unreviewed, and sometimes we may find assignment errors. There is more helpful information (on the left field), like the availability of a 3D structure, catalytic activity, etc.
We will choose BCHD_RHOCB (entry P26175), the subunit BchD from Rhodobacter capsulatus
. In plants, the homologous subunit is called ChlD
; "B" in BchD indicates that the protein is involved in bacteriochlorophyll
syntheses, while ChlD is related to chlorophyll synthesis.
On the page that will open after clicking the entry ID P26175
, we will find a lot of detailed information on magnesium chelatase
- its biological function (photosynthesis, magnesium chelatase activity), type of ligands/substrate it binds, catalytic function (insertion of magnesium and ATP hydrolysis), links to published works, links to entries related to this particular protein in other databases, and of course the amino acid sequence of BchD.
Clicking "Family & Domains
" on the menu on the left of the UniProt page will show the domain content of BchD. However, in this case, we only get the vWFA domain (von Willebrand Factor A-like domain superfamily
) at the C-terminal of the protein (residues 375-559). The InterPro database link also directs us to the BchD/ChlD, vWFA domain
page. It is nice to see that they reference our publication from 2001, Fodje et al., J. Mol. Biol. 311, 111-22
. We were the first to identify this domain in the magnesium chelatase family. A characteristic feature of the vWFA domain is the conservation of the so-called MIDAS motif. MIDAS stands for metal ion-dependent adhesion site (see a paper by Lacy et al. 2004 for a detailed description of the structure).
The conserved sequence consists of the DXSXS motif and additional threonine and aspartate (T and D) residues. The DXSXS motif residues in BchD are D385, S387 S389, which are close to the N-terminus of the vWFA domain, while threonine T452 and aspartate D482 are located further down in the sequence. The alignment in the image below (click to see it, it can also be found at the Conserved Protein Domain Family server
at NCBI) shows the position of these invariant residues (yellow boxes, also marked by a # on top of the alignment). These residues are involved in metal ion binding, Ca2+
. We still do not know the exact function of this domain within the magnesium chelatase complex.