The Structural Versatility of the BTB Domains of KCTD Proteins and Their Recognition of the GABAB Receptor

Abstract

Several recent investigations have demonstrated that members of the KCTD (Potassium Channel Tetramerization Domain) protein family are involved in fundamental processes. However, the paucity of structural data available on these proteins has frequently prevented the definition of their biochemical role(s). Fortunately, this scenario is rapidly changing as, in very recent years, several crystallographic structures have been reported. Although these investigations have provided very important insights into the function of KCTDs, they have also raised some puzzling issues. One is related to the observation that the BTB (broad-complex, tramtrack, and bric-à-brac) domain of these proteins presents a remarkable structural versatility, being able to adopt a variety of oligomeric states. To gain insights into this intriguing aspect, we performed extensive molecular dynamics simulations on several BTB domains of KCTD proteins in different oligomeric states (monomers, dimers, tetramers, and open/close pentamers). These studies indicate that KCTD-BTB domains are stable in the simulation timescales, even in their monomeric forms. Moreover, simulations also show that the dynamic behavior of open pentameric states is strictly related to their functional roles and that different KCTDs may form stable hetero-oligomers. Molecular dynamics (MD) simulations also provided a dynamic view of the complex formed by KCTD16 and the GABA_B2 receptor, whose structure has been recently reported. Finally, simulations carried out on the isolated fragment of the GABA_B2 receptor that binds KCTD16 indicate that it is able to assume the local conformation required for the binding to KCTD.

Keywords: molecular dynamics simulations; oligomerization; protein–protein interactions.

Figure 1

Cartoon representation (A) and topology (B) of the five α-helices and the three/four-stranded β-sheet motif of the BTB domain. Sequence alignment of the BTB domains of the KCTD proteins considered in this study (C). Residues belonging to α-helices and β-strands are colored in blue and red, respectively. For KCTD12, whose structure has not been experimentally determined, the assignment of the secondary structure is not reported on the sequence.

Figure 2

Structure stability of SHKBP1^BTB throughout the simulation: C^α-based RMSD values of trajectory structures calculated against the starting crystallographic model (A), time evolution of the secondary structure content (B). The RMSD values have been calculated on the whole structure (residues 18-120, black) or by excluding the last six residues at the C-terminus (residues 18-114, grey). Cartoon representation of SHKBP1^BTB (C), α-helices and β-strands are colored in blue and red, respectively. The last six residues at the C-terminus are in yellow. C^α-based RMSF values of residues of SHKBP1^BTB calculated in the 50–200 ns trajectory region (D). The protein secondary structure elements are reported as bars (α-helices in blue and β-strands in red).

Figure 3

Structure stability of KCTD5^BTB (A) and KCTD13^BTB (B) monomers throughout the MD simulations: C^α-based RMSD values of trajectory structures computed against the starting model, time evolution of the secondary structure content, C^α-based RMSF values computed in the equilibrated region of the trajectory (50–200 ns). The protein secondary structure elements are reported as bars (α-helices in blue and β-strands in red).

Figure 4

Structure stability of KCTD13^BTB tetramer throughout the MD simulation: C^α-based RMSD values of trajectory structures against the starting model (A), time evolution of the secondary structure content (B), C^α-based RMSF values computed in the 50–150 ns trajectory region (C). The protein secondary structure elements are reported as bars (α-helices in blue and β-strands in red).

Figure 5

C^α-based RMSD values of trajectory structures against the starting model obtained in the MD simulations performed on KCTD1^BTB and KCTD16^BTB open pentamers (A). Distance between the centers of mass of the two external domains that delimitate the gap in KCTD1^BTB and KCTD16^BTB open pentamers (B). The red dot indicates the common starting value (~39 Å). Superimposition of the C^α-trace of KCTD1^BTB and KCTD16^BTB average structures computed in the equilibrated region of trajectories (50–200 ns) (C). Distribution of the number of atoms of the two external domains that are within 6.0 Å in KCTD1^BTB (D) and KCTD16^BTB (E).

Figure 6

Average structure of KCTD12-16^BTB hetero-pentamer computed in the 50–150 ns region of the MD trajectory (A). Distance between the centers of mass of the two external domains that delimitate the gap (B). Distribution of the number of atoms of the two external domains that are within 6.0 Å (C).

Figure 7

Cartoon representation of the crystal structure of the complex between KCTD16^BTB and GABA_B2R peptide (PDB ID: 6M8R) used as starting model in the MD simulation. Time evolution of the distances between pairs of atoms involved in the formation of H-bonding interactions in the complex. The H-bonds between atoms Tyr36O^η-Gly908O, Gly33O-L894N, Gln34O^ε1-Leu896N, and Lys67O-Arg891N^η1 that are present in the starting crystallographic model are conserved throughout the simulation. The H-bonds between atoms Gln34^Oε1-His901^Nε2, Gly33^O-Gln895^N, and Gln34^Oε1-Gln895^N that are not present in the starting structure but are present in the structure of the complex between KCTD16^BTB and the GABA_B2R peptide encompassing residues 895-909 are formed in the simulation. Plots are colored to identify the chains of KCTD16^BTB that interact with the peptide (in grey).

Figure 8

Representation of the fully extended structure (φ = −120°, ψ = 130°, ω = 180°) (green) of the GABA_B2R peptide (residues 881-913) used as starting model in the MD simulation of the isolated peptide. The aminoacid sequence and the structure (grey) of the peptide extracted from the crystallographic complex with KCTD16^BTB (PDB ID: 6M8R) are also shown. Residues 885-890 and 899-903 which adopt a helical conformation are colored in blue in the sequence (A). Time evolution of the radius of gyration R_g (B) and of the secondary structure content (C) of GABA_B2R peptide. Representative examples of the conformational states detected in the simulation (green) superimposed to the crystallographic model (grey) in the regions 885-890 and 899-903 (D).