Figure 2. The ligand-binding modes of Rossman and P-loop proteins.

The phosphate binding loops (PBLs) of both lineages connect the C-terminus of β1 to the N-terminus of α1 (conserved glycine residues are colored magenta). The Rossmann β2-Asp, and the P-loop Walker B-Asp, are in green sticks. Water molecules are denoted by red spheres, and metal dications by green spheres. (A) The canonical P-loop NTPase binding mode. The phosphate binding loop (the P-loop Walker A motif; GxxGxGK(T/S)) begins with the first conserved Gly residue at the tip of β1 and ends with a Thr/Ser residing within α1. The Walker B-Asp, located at the tip of β3, interacts with the catalytic Mg²⁺, either directly or via a water molecule, as seen here. (B) The canonical Rossmann binding mode. The phosphate binding site includes a canonical water molecule (α1 has been rendered transparent so that the conserved water is visible). The Asp sidechain at the tip of β2 (β2-Asp) forms a bidentate interaction with both hydroxyls of the ribose. Note also the opposite orientations of the ribose and adenine moieties in P-loops (pointing away from the β-sheet) versus Rossmann (pointing towards the β-sheet). (C) Tubulin is a GTPase that belongs to the Rossmann lineage. It possesses the canonical Rossmann strand topology, phosphate binding loop (including the mediating water), and β2-Asp. However, the ligand, GTP, is bound in the P-loop NTPase mode (as in A). Accordingly, the β2-Asp makes a water mediated interaction with the catalytic metal cation (Ca²⁺or Mg²⁺) thus acting in effect as a Walker B-Asp (the metal cation’s coordination schemes are also identical, see Figure 2—figure supplement 3). ECOD domains used in this figure, from left to right, are e1yrbA1, e1lssA1, and e5j2tB1. All structure figures were prepared in PyMOL (pymol.org).

Figure 2—figure supplement 1. Rossmann domain binding ATP in the canonical binding mode.

The phosphate binding loop of ECOD domain e3h5nA5, a Rossmann domain from the ECOD F-group 2003.1.9.15, binds the nucleotide ATP in a largely canonical fashion, including the bidentate interaction between the β2-Asp and the ribose hydroxyls (see Main Text). The conserved water characteristic of the Rossmann fold is shown as a red sphere. An Mg²⁺ cation is shown as a green sphere. Other F-groups with canonical binding of an NTP include 2003.1.9.10 and 2003.1.4.3.

Figure 2—figure supplement 2. Features of the tubulin binding site (see also Supplementary file 1).

(A) The tubulin phosphate binding loop resides between β1 and α1 (cyan; ECOD domain e5j2tA1) as in all Rossmanns, yet is shorter and more compact than the canonical Rossmann binding loop (yellow; ECOD domain e1lssA1). (B) The conserved β2-Asp (yellow structures; ECOD domains e1ffxB2, e1sa1C2, e2btoA2, e2hxfA2, e3cb2A2, e3e22C2, e3r4vA1, e3zbqA2, e4ffbA4, e4ffbB1) is replaced by asparagine in some structures (cyan structures; ECOD domains e1rq2A2, e1w5fA2, e2r6r11, e2vamA1, e2vapA2, e2xkbC5, e3v3tA3, e3zidA1, e4b45A3, e4b46A1, e4dxdA1, e4e6eA1, e4m8iA2). This residue is positioned to interact with the catalytic Mg²⁺ (green spheres), typically via a water molecule (red spheres).

Figure 2—figure supplement 3. The tubulin β2-Asp and the P-loop Walker B interact with waters that occupy equivalent sites around the catalytic Mg²⁺ cation.

The catalytic Mg²⁺ cation in both tubulin (top panel) and P-loop domains (bottom panel) forms octahedral coordination complexes. Both the β2-Asp of tubulin and the Walker B of P-loop interact with the water at position 6 of the coordination sphere. Water molecules are rendered as red spheres and Mg²⁺ cations are rendered as green spheres.