Structural insights into Charcot–Marie–Tooth disease‐linked mutations in human GDAP1

Abstract

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral polyneuropathy in humans, and its different subtypes are linked to mutations in dozens of different genes. Mutations in gangliosideinduced differentiation-associated protein 1 (GDAP1) cause two types of CMT, demyelinating CMT4A and axonal CMT2K. The GDAP1-linked CMT genotypes are mainly missense point mutations. Despite clinical profiling and in vivo studies on the mutations, the etiology of GDAP1-linked CMT is poorly understood. Here, we describe the biochemical and structural properties of the Finnish founding CMT2K mutation H123R and CMT2K-linked R120W, both of which are autosomal dominant mutations. The disease variant proteins retain close to normal structure and solution behavior, but both present a significant decrease in thermal stability. Using GDAP1 variant crystal structures, we identify a side-chain interaction network between helices ⍺3, ⍺6, and ⍺7, which is affected by CMT mutations, as well as a hinge in the long helix ⍺6, which is linked to structural flexibility. Structural analysis of GDAP1 indicates that CMT may arise from disruption of specific intra-and intermolecular interaction networks, leading to alterations in GDAP1 structure and stability, and, eventually, insufficient motor and sensory neuron function.Inherited polyneuropathies are a genetically and clinically diverse group of neurodegenerative diseases affecting motor and sensory neurons in the peripheral nervous system (PNS) [1,2]. Mutations in dozens of genes expressed in the PNS cause Charcot-Marie-Tooth disease (CMT). Based on clinical findings, CMT can be classified into three forms: demyelinating, axonal, and intermediate [3,4]. The progress of CMT is linked to the hereditary pattern, whereby the autosomal recessive form has an earlier onset and more severe symptoms than the autosomal dominant form [5][6][7]. Understanding the molecular function of the proteins involved in the etiology of neuropathies is vital in efforts toward treatment and diagnosis.

Conserved intramolecular networks in GDAP1 are closely connected to CMT-linked mutations and protein stability

“…Building upon earlier work on GDAP1 structure [10–12], we focused here on several CMT-linked variants that reside on different secondary structure elements on the GDAP1 crystal structure. While we earlier specifically looked at R120W and H123R on helix α3 [12], here we produced and characterised the variants R120Q, R161H, A247V, H256R, R282H, R310Q, and R310W. The stability and solution structure were studied for all variants, while crystal structures were determined for three of them: R120Q, A247V, and R282H.…”

“…The majority of CMT-linked missense mutations in GDAP1 are located within the vicinity of the hydrophobic clusters of the GST-like domains and the dimer interface [11], and the variants may induce changes in intramolecular hydrogen bonding networks [12]. In addition to the R120W and H123R studied earlier [12], we now determined three new mutant crystal structures: R120Q, A247V, and R282H. These mutations reside in helices α3 (R120Q), α7 (A247V), and α8 (R282H), which are core elements of the GDAP1 fold.…”

“…The two models were used to shed light on conformational changes and sequence conservation in GDAP1. Earlier structure-based bioinformatics results [12] were analysed further with respect to the mutational spectrum of GDAP1.…”

“…GDAP1 is constructed of two GST-like domains in the N and C terminus, followed by a transmembrane helix, which anchors the protein into the MOM. Structural data have shown a covalently bound dimer interface in GDAP1 [11, 12], and while dimerization is a common feature in catalytic GSTs [13], the GDAP1 dimer is formed differently [11]. Enzymatic activity of GDAP1 has not been convincingly demonstrated, nor has any substrate been identified in vivo.…”

The GDAP1 p.Glu222Lys Variant-Weak Pathogenic Effect, Cumulative Effect of Weak Sequence Variants, or Synergy of Both Factors?

“…Not only do the various sequence variants contribute to the phenotype, but genetic interaction between two alleles of GDAP1 is important for the genetic load. Even the variant p.Glu222Lys seems to be weak in recent studies studying the interaction of the amino-acid GDAP1 residues, and the authors showed that the Glu222Lys residue is sandwiched between three Arg side chains (Arg120, Arg225, Arg226) and Tyr124, and, additionally, this residue has van der Waals contacts to Leu239, the amino acid residue most frequently substituted in CMT4A [ 24 ]. This implies the importance of Glu222 amino acid residue in maintaining the proper structure of GDAP1 protein, and importantly its substitution can affect Leu239Phe residue.…”