The Domain-Specific and Temperature-Dependent Protein Misfolding Phenotype of Variant Medium-Chain acyl-CoA Dehydrogenase

The implementation of expanded newborn screening programs reduced mortality and morbidity in medium-chain acyl-CoA dehydrogenase deficiency (MCADD) caused by mutations in the ACADM gene. However, the disease is still potentially fatal. Missense induced MCADD is a protein misfolding disease with a molecular loss-of-function phenotype. Here we established a comprehensive experimental setup to analyze the structural consequences of eight ACADM missense mutations (p. Ala52Val, p. Tyr67His, p. Tyr158His, p. Arg206Cys, p. Asp266Gly, p. Lys329Glu, p. Arg334Lys, p. Arg413Ser) identified after newborn screening and linked the corresponding protein misfolding phenotype to the site of side-chain replacement with respect to the domain. With fever being the crucial risk factor for metabolic decompensation of patients with MCADD, special emphasis was put on the analysis of structural and functional derangements related to thermal stress. Based on protein conformation, thermal stability and kinetic stability, the molecular phenotype in MCADD depends on the structural region that is affected by missense-induced conformational changes with the central beta-domain being particularly prone to structural derangement and destabilization. Since systematic classification of conformational derangements induced by ACADM mutations may be a helpful tool in assessing the clinical risk of patients, we scored the misfolding phenotype of the variants in comparison to p. Lys329Glu (K304E),the classical severe mutation, and p. Tyr67His (Y42H),discussed to be mild. Experiments assessing the impact of thermal stress revealed that mutations in the ACADM gene lower the temperature threshold at which MCAD loss-of-function occurs. Consequently, increased temperature as it occurs during intercurrent infections, significantly increases the risk of further conformational derangement and loss of function of the MCAD enzyme explaining the life-threatening clinical courses observed during fever episodes. Early and aggressive antipyretic treatment thus may be life-saving in patients suffering from MCADD

Protein misfolding is the molecular mechanism underlying MCADD identified in newborn screening

Newborn screening (NBS) for medium-chain acyl-CoA dehydrogenase deficiency (MCADD) revealed a higher birth prevalence and genotypic variability than previously estimated, including numerous novel missense mutations in the ACADM gene. On average, these mutations are associated with milder biochemical phenotypes raising the question about their pathogenic relevance. In this study, we analyzed the impact of 10 ACADM mutations identified in NBS (A27V, Y42H, Y133H, R181C, R223G, D241G, K304E, R309K, I331T and R388S) on conformation, stability and enzyme kinetics of the corresponding proteins. Partial to total rescue of aggregation by co-overexpression of GroESL indicated protein misfolding. This was confirmed by accelerated thermal unfolding in all variants, as well as decreased proteolytic stability and accelerated thermal inactivation in most variants. Catalytic function varied from high residual activity to markedly decreased activity or substrate affinity. Mutations mapping to the β-domain of the protein predisposed to severe destabilization. In silico structural analyses of the affected amino acid residues revealed involvement in functionally relevant networks. Taken together, our results substantiate the hypothesis of protein misfolding with loss-of-function being the common molecular basis in MCADD. Moreover, considerable structural alterations in all analyzed variants do not support the view that novel mutations found in NBS bear a lower risk of metabolic decompensation than that associated with mutations detected in clinically ascertained patients. Finally, the detailed insight into how ACADM missense mutations induce loss of MCAD function may provide guidance for risk assessment and counseling of patients, and in future may assist delineation of novel pharmacological strategies

Secondary structure analysis (CD spectroscopy).

<p>Secondary structure analysis (CD spectroscopy).</p

Localization of side chains affected by mutations and residual enzyme function of MCAD variants.

<p>(A) Mutations in the <i>ACADM</i> gene map to side chains (red) of all regions of the MCAD monomer. The mature MCAD protein is shown and numbering of side chains refers to the primary sequence after cleavage of an N-terminal 25 amino acids mitochondrial targeting peptide (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093852#pone-0093852-t001" target="_blank">Table 1</a>). Side chains, FAD cofactor (yellow) and the substrate analogue 3-thiaoctanoyl-CoA (orange) are shown as stick models. (B, C) Structural localization of affected side-chains at the surface of subunit A (gray) relative to the other subunits (B, green; C pink; D, blue) in the MCAD tetramer. (D) Surface view of the MCAD tetramer with 3-thiaoctanoyl-CoA (orange) bound to the substrate binding site and the positioning of FAD (yellow) relative to subunits A (gray), B (green), and D (blue). (E) Relative enzyme activity of wild-type and variant MCAD proteins. Specific activity was determined using purified recombinant protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093852#pone.0093852-Maier2" target="_blank">[16]</a> and residual activity was measured upon expression in COS-7 cells. Data are given as means and SD of three replicates.</p

Mean transition midpoints of thermal denaturation (CD spectroscopy, FAD fluorescence, ANS fluorescence).

<p>Mean transition midpoints of thermal denaturation (CD spectroscopy, FAD fluorescence, ANS fluorescence).</p

Molecular phenotypes of thermal, kinetic, and conformational stability.

<p>Data derived from experiments depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093852#pone-0093852-g002" target="_blank">Figures 2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093852#pone-0093852-g003" target="_blank">3</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093852#pone-0093852-g005" target="_blank">5</a> were combined and visualized as 3D plot. Thermal stability (y-axis) refers to midpoints of thermal denaturation determined by CD spectroscopy, FAD-DSF, and ANS-DSF. Kinetic stability (x-axis) refers to the E_A determined by thermal inactivation experiments. Conformational stability (z-axis) refers to secondary structure (CD spectra), hydrophobicity (ANS fluorescence), and FAD binding capacity (intrinsic FAD fluorescence) in the absence of heat-induced stress.</p

Mutations.

<p>Mutations.</p

Missense mutations induce conformational alterations of variant MCAD proteins.

<p>(A) The composition of secondary structure elements of wild-type and variant MCAD in the absence of thermal stress was determined by circular dichroism applying the CD spectra deconvolution software CDNN <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093852#pone.0093852-Bhm1" target="_blank">[23]</a>. The spectra showed typical secondary structure characteristics of proteins with predominating α-helical content. (B) Hydrophobicity of wild-type and variant MCAD proteins in the absence of thermal stress was determined using the fluorescent dye ANS. An increase in the fluorescence signal points to increased binding of the ANS dye to hydrophobic groups. Data represent means and SEM of three independent experiments. (C) Structural integrity of wild-type and variant MCAD proteins in the absence of thermal stress was determined by intrinsic FAD fluorescence. An increase in the fluorescence signal points to a release of FAD from the MCAD protein. Data represent means and SEM of three independent experiments. (B, C) Data are grouped according to the domain affected by mutations (αD_N, N-terminal α-helical domain; βD, β-sheet domain; αD_C, C-terminal α-helical domain).</p

Kinetics of thermal inactivation.

<p>(A) Kinetics of thermal inactivation experiments probing dynamics of heat-induced loss of function. Wild-type and variant MCAD proteins were incubated at indicated temperatures and residual activity was plotted as a function of incubation time. (B) Arrhenius Plots resulting from kinetic constants <i>k</i> of time-dependent thermal denaturation experiments. (C) Activation energies (E_A) calculated from Arrhenius Plots reflect the energy barrier between the conformational state with full residual activity and the non-active state after application of thermal stress.</p

Mutations of the middle β-domain of the MCAD protein are associated with accelerated heat-induced unfolding.

<p>(A) Thermal stress-induced denaturation monitored by circular dichroism (CD) probing loss of secondary structure. Denaturation profiles of the fractional change of the α-helical secondary structures of wild-type and variant MCAD were measured at 208 nm. Data are given as non-linear fit of the mean of three independent experiments where <i>1</i> represents the native state and <i>0</i> the unfolded state. (B) Thermal stress-induced denaturation monitored by FAD differential scanning fluorimetry (DSF) probing FAD binding capacity. The temperature dependent release of intrinsic FAD of wild-type and variant MCAD was measured at 530 nm (excitation 450 nm). Data are given as non-linear fit of the mean of three independent experiments where <i>0</i> represents the native state and <i>1</i> the unfolded state. (C) Thermal stress-induced denaturation monitored by ANS-DSF probing global unfolding. Unfolding profiles of wild-type and variant MCAD were determined as changes in ANS fluorescence emission at 450 nm (excitation 395 nm). All unfolding patterns revealed a biphasic unfolding mechanism with a distinct low- and high-temperature transition, depicted in two red lines in the unfolding curve of wild-type MCAD. Data are grouped according to the domain affected by mutations (αD_N, N-terminal α-helical domain; βD, β-sheet domain; αD_C, C-terminal α-helical domain) and given as mean of three independent experiments where <i>0</i> represents the native state and <i>1</i> the unfolded state.</p