Structure of the human transcobalamin beta domain in four distinct states

Vitamin B12 (cyanocobalamin, CNCbl) is an essential cofactor-precursor for two biochemical reactions in humans. When ingested, cobalamins (Cbl) are transported via a multistep transport system into the bloodstream, where the soluble protein transcobalamin (TC) binds Cbl and the complex is taken up into the cells via receptor mediated endocytosis. Crystal structures of TC in complex with CNCbl have been solved previously. However, the initial steps of holo-TC assembly have remained elusive. Here, we present four crystal structures of the beta domain of human TC (TC-beta) in different substrate-bound states. These include the apo and CNCbl-bound states, providing insight into the early steps of holo-TC assembly. We found that in vitro assembly of TC-alpha and TC-beta to a complex was Cbl-dependent. We also determined the structure of TC-beta in complex with cobinamide (Cbi), an alternative substrate, shedding light on the specificity of TC. We finally determined the structure of TC-beta in complex with an inhibitory antivitamin B12 (anti-B12). We used this structure to model the binding of anti-B12 into full-length holo-TC and could rule out that the inhibitory function of anti-B12 was based on an inability to form a functional complex with TC.

The table below summarises the geometric issues observed across the polymeric chains and their t to the electron density. The red, orange, yellow and green segments on the lower bar indicate the fraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria. A grey segment represents the fraction of residues that are not modelled. The numeric value for each fraction is indicated below the corresponding segment, with a dot representing fractions <=5% The upper red bar (where present) indicates the fraction of residues that have poor t to the electron density. The numeric value is given above the bar.

Mol Chain Length
Quality of chain 1 A 108 The following table lists non-polymeric compounds, carbohydrate monomers and non-standard residues in protein, DNA, RNA chains that are outliers for geometric or electron-density-t criteria: Mol Type Chain Res Chirality Geometry Clashes Electron density 2 CBY A 501 ---X O There are 4 unique types of molecules in this entry. The entry contains 2075 atoms, of which 1000 are hydrogens and 0 are deuteriums.
In the tables below, the ZeroOcc column contains the number of atoms modelled with zero occupancy, the AltConf column contains the number of residues with at least one atom in alternate conformation and the Trace column contains the number of residues modelled with at most 2 atoms.
Mol Chain Residues Atoms ZeroOcc AltConf Trace O These plots are drawn for all protein, RNA and DNA chains in the entry. The rst graphic for a chain summarises the proportions of the various outlier classes displayed in the second graphic. The second graphic shows the sequence view annotated by issues in geometry and electron density. Residues are color-coded according to the number of geometric quality criteria for which they contain at least one outlier: green = 0, yellow = 1, orange = 2 and red = 3 or more. A red dot above a residue indicates a poor t to the electron density (RSRZ > 2). Stretches of 2 or more consecutive residues without any outlier are shown as a green connector. Residues present in the sample, but not in the model, are shown in grey.
• Molecule 1: Transcobalamin-2 Chain A: The Z score for a bond length (or angle) is the number of standard deviations the observed value is removed from the expected value. A bond length (or angle) with |Z| > 5 is considered an outlier worth inspection. RMSZ is the root-mean-square of all Z scores of the bond lengths (or angles).

Mol Chain Bond lengths Bond angles
There are no bond length outliers.
There are no bond angle outliers.
There are no chirality outliers.
There are no planarity outliers. The all-atom clashscore is dened as the number of clashes found per 1000 atoms (including hydrogen atoms). The all-atom clashscore for this structure is 16.
All (32) close contacts within the same asymmetric unit are listed below, sorted by their clash magnitude. In the following table, the Percentiles column shows the percent Ramachandran outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.
The Analysed column shows the number of residues for which the backbone conformation was analysed, and the total number of residues.
Mol Chain Analysed Favoured Allowed Outliers Percentiles 1 A 108/108 (100%) 104 (96%) 3 (3%) 1 (1%) 20 3 All (1)  In the following table, the Percentiles column shows the percent sidechain outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.
The Analysed column shows the number of residues for which the sidechain conformation was analysed, and the total number of residues.  (and percentage) of RSRZ outliers, followed by percent RSRZ outliers for the chain as percentile scores relative to all X-ray entries and entries of similar resolution. The OWAB column contains the minimum, median, 95 th percentile and maximum values of the occupancy-weighted average B-factor per residue. The column labelled`Q< 0.9' lists the number of (and percentage) of residues with an average occupancy less than 0.9.