Phenotypic spectrum of the tubulin-related disorders and functional implications of disease-causing mutations

Abstract

A spectrum of neurological disorders characterized by abnormal neuronal migration, differentiation, and axon guidance and maintenance have recently been attributed to missense and splice-site mutations in the genes that encode α-tubulin and β-tubulin isotypes TUBA1A, TUBA8, TUBB2B, and TUBB3, all of which putatively coassemble into neuronal microtubules. The resulting nervous system malformations can include different types of cortical malformations, defects in commissural fiber tracts, and degeneration of motor and sensory axons. Many clinical phenotypes and brain malformations are shared among the various mutations regardless of structural location and/or isotype, while others segregate with distinct amino acids or functional domains within tubulin. Collectively, these disorders provide novel paradigms for understanding the biological functions of microtubules and their core components in normal health and disease.

Figure 1. Five functional domains of tubulin

Tubulin is comprised of three separate functional domains that participate in heterodimer stability, longitudinal and lateral protofilament interactions, nucleotide exchange and hydrolysis, and microtubule-protein interactions. (A) α–and β–tubulin each contain a GTP binding pocket (black) at their N and E-sites (1 and 2, respectively), found within the N-terminal domain of the protein. Nucleotide binding is essential for protein folding, heterodimer stability, and microtubule dynamics. The N-site is formed primarily by α-tubulin residues (1, black) and is stabilized by interactions with some β-tubulin residues (not shown). GTP (1, orange) bound to α–tubulin is non-exchangeable because the N-site is buried at the intra-heterodimer interface. The E-site is formed primarily by residues located near the plus-end of β-tubulin (2, black), and also interacts with some α-tubulin residues located at the inter-heterodimer interface (not shown). GTP bound to β-tubulin is eventually hydrolyzed to GDP (2, orange) once heterodimers have incorporated into microtubules, and the energy released causes microtubule disassembly due to protofilament curling (depicted in figure 3). This force is countered by lateral protofilament interactions and the abundance of GTP-bound β-tubulin heterodimers at the microtubule plus-end. (B) Residues found between the N-terminal and intermediate domains of tubulin (yellow) help regulate the stability of the tubulin heterodimer and structural rearrangements coupled to GTP hydrolysis. (C) Longitudinal interactions are mediated by a series of highly conserved residues found at the interfaces between the inter- and intra-heterodimer (light blue), and facilitate the stability of tubulin heterodimers and the assembly of longitudinal protofilaments (cartoon on left). (D) Lateral protofilament interactions are facilitated by residues that flank the sides and inner surface of heterodimers (purple), and are important for “zipping up” the open sheet of longitudinal protofilaments into a hollow tube (cartoon on left). Lateral protofilament interactions also regulate microtubule dynamics following GTP hydrolysis. (E) MAP and motor protein interactions are mediated by external α–helices and adjacent grooves on the outer surface of the microtubule (green). Cartoon on the left depicts motor protein movement and MAP binding along the surface of a microtubule. 3D tubulin schematics were generated using PyMol (PDB: 1JFF). Cartoons labeled “MT” in panels B–E depict the orientation of heterodimers relative to the assembled microtubule. Transparent tubulin in panels C and D are approximations of longitudinal and lateral interactions, respectively, showing adjacent heterodimer subunits within microtubules.

Figure 2. Three-dimensional mapping of disease-causing amino acid substitutions in TUBB2B, TUBB3, and TUBA1A

Functional domains of tubulin are colored as per Figure 1A–E, and residues altered by mutations are highlighted in red. Row 1: Panel depicting mutations altering residues located in the GTP binding pocket, and regions of heterodimer stability, and longitudinal and lateral interactions. “Side views” are shown for each tubulin, and an additional “longitudinal” view is shown for TUBA1A. Row 2: Panel depicting mutations altering residues located in regions of MAP/motor protein interactions. “Topdown” view onto the external helices of tubulin is shown. (A, TUBB2B: PMG) Substitutions in TUBB2B that cause polymicrogyria alter amino acids in domains important for GTP binding, heterodimer stability, and longitudinal interactions (row 1), but are not located in regions that mediate lateral interactions (row 1) or MAP/Motor protein binding (row 2). (A, TUBB3: MCD) Substitutions in TUBB3 that cause gyral malformations are located primarily in rgions that regulate GTP binding, heterodimer stability, and longitudinal and lateral interactions (row 1). Exceptions are M388 and A302 (row 2). A302 is located within a loop that could be important for both heterodimer stability and MAP/motor protein interactions. Similarly, M388 could also regulate MAP/motor protein interactions, and is in proximity to residues at the plus-end of β-tubulin that mediate inter-heterodimer contacts. (A, TUBB3: CFEOM3) Substitutions in TUBB3 that cause axon guidance defects and CFEOM3 are largely found in regions of MAP/motor protein interactions (row 2). The exceptions are R62 and A302, the former of which is located in a loop mediating lateral interactions. (B, TUBA1A: LIS to MCD) Substitutions in TUBA1A are located in all major functional domains. PMG = polymicrogyria, MCD = malformations of cortical development, CFEOM3 = congenital fibrosis of the extraocular muscles type 3, LIS = lissencephaly.

Figure 3. Summary depicting the proposed functional effects of disease-causing mutations in α–and β-tubulin

(A) Overview of the tubulin heterodimer folding pathway. (A) (1) Nascent tubulin polypeptides are delivered to the cytosolic chaperone (CCT) in order to generate folding intermediates with GTP binding pockets. (2) α–and β-tubulin folding intermediates are then released, bound, and stabilized by a second set of chaperones, TBCA and TBCD (β-tubulin) and TBCB and TBCE (α–tubulin). (3) TBCD and TBCE form a complex to co-assemble the tubulin heterodimer, and bind TBCC. (4) This triggers the hydrolysis of GTP bound to β-tubulin and releases the tubulin heterodimer from the folding complex. (5) Following the exchange of GDP for GTP in β-tubulin, the heterodimers are capable of incorporating into microtubules. Mutations in tubulin are predicted to diminish the levels of functional tubulin heterodimers by disrupting the formation of the GTP binding pocket and/or interactions with protein chaperones. (B) Tubulin heterodimers assemble in a head to tail fashion to form a sheet of longitudinal protofilaments at the growing plus-end of a microtubule. Lateral interactions between adjacent protofilaments cause the open sheet to close and assemble into a hollow tube. (C) Mutations in α–and β-tubulin found at inter-heterodimer interfaces and/or regions of lateral protofilament interactions are predicted to impede the polymerization and dynamic properties of microtubules, resulting in microtubules that may be (1) relatively non-dynamic with reduced frequencies of growth and shortening, or (2) unstable and more likely to depolymerize. (D) The external microtubule surface interacts with kinesin and dynein motors, allowing the anterograde and retrograde transport of proteins and organelles along microtubules. MAPs also bind to the external surface and extrinsically regulate microtubule stability and dynamics. Mutations found in the external helices of tubulin are demonstrated or predicted to alter these types of protein interactions.