BACKGROUND
multiple acyl-coa dehydrogenase deficiency , also known as glutaric aciduria type ii, is an autosomal recessively inherited disorder of fatty acid metabolism  <cit> .
in the majority of cases, the disorder is caused by a defect in either the alpha- or beta-subunit of the electron transfer flavoprotein  or the electron transfer flavoprotein dehydrogenase  genes.
however, in some patients, the disorder may result from a few, currently unidentified, disruptions to riboflavin metabolism  <cit> .
because of a deficiency of etf or etfdh, the acyl-coa dehydrogenases are unable to transfer the electrons generated by the dehydrogenation reactions.
this results in the accumulation of various intramitochondrial acyl-coa esters.
recently, etfdh mutations were reported to be major causes of riboflavin-responsive madd  <cit> .
characteristics such as organic aciduria may be detected only during periods of illness or catabolic stress.
in the majority of cases, depending on the metabolic status of the patient, plasma acylcarnitine analysis may not provide a definitive diagnosis.
amongst the most daunting challenges facing madd is metabolic heterogeneity, in particular, for the diagnosis of madd.
thus, numerous molecular diagnostic methods have been established for analyzing the etf and/or etfdh genes for the purpose of achieving the final diagnosis of madd.
previously, we reconfirmed the high prevalence of the c.250g > a  mutation in taiwanese patients with riboflavin-responsive madd, as well as in the normal population using a high-resolution melting  analysis  <cit> .
it should be noted that all the nine patients examined, including three pairs of siblings, harbor this mutation and four were homozygous.
we estimated the allele frequency to be  <dig>  for the p.ala84thr mutant .
thus, the estimated carrier frequency of this mutation in the taiwanese population is 1: <dig> .
the homozygous mutant frequency was 1: <dig> .
nevertheless, the carrier frequency of c.250g > a is estimated to be  <dig> % amongst the population in southern china.
thus, ~1: <dig>  han chinese are expected to suffer from riboflavin-responsive madd  <cit> .
intriguingly, p.ala84thr has been identified in asian countries including taiwan, hong kong, singapore, thailand and southern china  <cit> .
a total of  <dig> patients were examined from the above referenced literature and diagnosed with late-onset madd.
amongst these patients, the mutation c.250g > a in the etfdh gene was detected in  <dig> patients with homozygous mutations and  <dig> patients with heterozygous mutations.
to date, this mutation has never been reported in western countries  <cit> .
thus, it has been suggested that the most common mutation p.ala84thr is ethnic-specific.
in addition, the mutation of p.ala84thr most likely accounts for the mild phenotype and response to riboflavin.
here, a patient presented at 11/ <dig> years represented a mild to late-onset madd and the patient slightly improved following riboflavin treatment .
the patient had compound heterozygous mutations  of the etfdh gene.
currently, there is no related study to explore the underlying mechanism for the most common mutation .
flavin adenine dinucleotide  is a cofactor for the electron transfer flavoprotein:ubiquinone oxidoreductase  which constitutes the electron-transport pathway for a number of mitochondrial flavoprotein dehydrogenases that are mainly involved in fatty-acid and amino-acid metabolism.
riboflavin  treatment has been shown to strikingly ameliorate the symptoms and metabolic profiles in madd patients  <cit> .
this may enhance the conformational stabilization of the mutant etf:qo protein  <cit> .
two of the mutations  are located in the fad-binding domain; however, the two amino acids do not have direct interactions with fad according to the predicted 3d structure of etf:qo.
the closest distances between the mutations  and the fad binding residues are  <dig>  and  <dig>  Å, respectively.
therefore, to explore the effects of the mutations on etf:qo dynamics, molecular dynamics  simulations of the wild type  and mutant type  etf:qo in the same model environment were compared.
besides the md simulations, an alternative method, normal mode analysis , for studying protein motions was used to analyze the dynamic correlations between the mutation sites and the fad-binding motif.
in this study, molecular modeling, md simulations and nma were performed to investigate the structural implications of the underlying mutations on the protein conformation.
the obtained results may provide an insight into the pathomechanism of madd.
methods
patients
the patient was a girl aged 11/ <dig> years who was clinically diagnosed with madd in  <dig>  her dried-blood spot sample was sent for acylcarnitine analysis.
the results demonstrated elevation of acylcarnitines during the illness, consistent with glutaric aciduria type  <dig>  gas chromatography-mass spectrometry revealed significant elevations of urinary glutaric acid, 3-methylglutarate, 3-methylcluconate, adipate, 3-methyladipate, octenedioate, suberate, and hexanoylglycine.
the present study was approved by the institutional review board  of kaohsiung medical university hospital.
genetic analysis
the blood samples were collected from the patient and the family members as well as from normal controls with informed consents.
the genomic dna samples were extracted from peripheral whole blood using the nucleospin® blood kit , according to the manufacturer's instructions.
identification of mutations
hrm analysis was applied for screening the mutation of etfdh, as previously described  <cit> .
pcr was carried out in duplicate in a  <dig> μl final volume using the lightcycler ®  <dig> high-resolution melting master   <dig> × buffer which contained taq polymerase, nucleotides, the dye resolight and  <dig> ng dna.
the primers and mgcl <dig> were used at a concentration of  <dig>  μm and  <dig>  mm, respectively for identifying the etfdh gene mutations.
the hrm assays were conducted using the lightcycler®  <dig> instrument  provided with the software lightcycler®  <dig> gene-scanning software version  <dig>  .
the pcr program required a sybr green i filter .
the program consisted of an initial denaturation-activation step at 95°c for  <dig> min, followed by a 45-cycle program.
the 45-cycle program consisted of the following steps: denaturation at 95°c for  <dig> s, annealing at 58°c or 60°c for  <dig> s and elongation at 72°c for  <dig> s with the reading of the fluorescence.
the acquisition mode was single.
the melting program consisted of three steps: denaturation at 95°c for  <dig> min, renaturation at 40°c for  <dig> min, and the subsequent melting that included continuous fluorescent readings from  <dig> to 90°c at a rate of  <dig> acquisitions per degree centigrade.
to confirm the results of the hrm analysis, a sequencing analysis was also performed for all the samples.
after the hrm analysis, the samples were purified using the favorprep™ pcr clean-up mini kit.
the pcr products generated after hrm were sequenced directly.
the sequence reaction was performed in a final volume of  <dig> μl, comprising  <dig> μl of the purified pcr product,  <dig>  μm of each pcr primer and  <dig> μl of the abi prism terminator cycle sequencing kit v <dig>  .
the sequencing program started from 96°c for  <dig> min and then followed by 25-cycle pcr program.
the program consisted of the following steps: denaturation at 96°c for  <dig> s; annealing at 50°c for  <dig> s; and elongation at 60°c for  <dig> min.
the sequence detection was performed using the abi prism  <dig> genetic analyzer .
to ensure that the mutation found in the patient was the one specifically present in patients with madd,  <dig> healthy control chromosomes were also screened.
sorting intolerant from tolerant  analysis
sift tool  <cit>  generates multiple alignments of the sequences over different species to look at the conserved sequence of a gene; it asses the conserved amino acid positions and analyzes the effect of missense changes on the conserved structure of proteins over the course of evolution.
the sift tool assigns a score to the mutations, and the score of <  <dig>  is considered potentially damaging.
structure prediction of human wild type  and mutant type  etf:qo
three-dimensional  structures of the human etf:qo were predicted using the  <dig> server  <cit> .
<dig> automatically uses an effective consensus strategy.
the above mentioned strategy combines structural-  <cit>  and profile-based  <cit>  comparison methods for both template selection and target-template alignment.
<dig> server selected the porcine etf:qo   <cit>  as the model template.
the etf:qo models were built based on a sequence and structural alignment from the above mentioned template.
a total of six models were yielded for each target by using a set of parameters.
these models were based on the alternative target-template alignments.
the qualities of the modeled structures were checked using ramachandran plots  <cit> .
the model with the highest quality was selected as the final predicted structure for the target.
finally, the protein-ligand complex was constructed by superimposing the predicted human etf:qo model to the 3d structure of the porcine etf:qo-ligand complex.
molecular dynamics simulations
molecular dynamics  simulations were performed using the software gromacs ver.
<dig> .
<dig>  <cit> .
opls-aa all-atom force field was used for the energy calculations.
the models were solvated with simple point charge  water molecules, and the models were simulated in a triclinic box  <cit>  with periodic boundary conditions.
the simulations were performed in the canonical nvt  ensemble.
the energy of the models was first minimized using the steepest descent algorithm.
this was followed by the position-restrained md simulations for  <dig> ps.
the linear constraint solver  algorithm was used to constrain the bonds  <cit> .
the md simulations were carried out at constant pressure and temperature for  <dig>  ns using an integration time step of  <dig> fs.
the non-bonded interactions were cutoff at  <dig> Å. at every  <dig> time steps, the coordinates from the md simulations were saved.
analysis of md trajectories
the objective was to understand the structural and functional implications of the amino acid substitution.
thus, the trajectories of wt and mt were analyzed for the following structural properties as a function of time: a) the root mean square deviation  of the cα atoms with respect to the starting conformation; b) rmsd of the fad-binding motif with respect to the starting conformation; and c) the b-factors  <cit>  of the cα atoms which were calculated from the last  <dig> ns of the md trajectories.
the b-factors in protein structures reflect the fluctuation of atoms about their average positions.
a large b-factor indicates high flexibility of individual atoms.
normal mode analysis
normal mode analysis  is a powerful tool to estimate the dynamics based on structure  <cit> .
low-frequency normal modes describing the large-scale real-world motions of a protein have been demonstrated to be related to biological function  <cit> .
in the present study, the nma was performed using the software gromacs ver.
<dig> .
<dig> by diagonalization of the mass-weighted hessian matrix.
the force field-based nma requires careful energy minimization of the initial structure.
we first minimized the human etf:qo structure with the steepest descent integrator for a couple of steps.
next, the l-bfgs integrator was used to minimize the maximum force close to zero and subsequently generate a hessian matrix.
the nma was performed using the algorithm in gromacs.
finally, the pair-wise residue correlation was calculated.
the above mentioned correlation was calculated as the average correlation summed over  <dig> lowest nonzero frequency normal modes  <cit> .
RESULTS
patient and molecular diagnostic
hrm analysis of etfdh gene revealed a novel t > c point mutation at position  <dig>  the point mutations were confirmed by direct dna sequencing .
the hotspot mutation c.250g > a   and a novel mutation c.383t > c   in the etfdh gene were identified in this patient.
these mutations are highly conserved amongst the different species .
the sift tool analysis revealed a score of <  <dig>  and predicted that the replaced amino-acid was potentially damaging.
this indicates that these mutations are most likely pathogenic.
figures 1e and 1f show the melting profiles of the etfdh mutations in the patient's family.
the patient had compound heterozygous mutations .
the sibling was a carrier of c.383t > c .
the father and mother were carriers of c.383t > c  in exon  <dig> and c.250g > a  in exon  <dig>  respectively .
the above mentioned mutation was not detected in the  <dig> unrelated control chromosomes  .
the pedigree is presented in figure 1h.
structure prediction of human etf:qo
human etf:qo models were constructed using the published porcine etf:qo crystal structure  as the template .
a close-up view of the hydrophobic residues which are located at the fad-binding domain, including v <dig>  i <dig>  a <dig>  v <dig>  l <dig>  v <dig>  l <dig>  and f <dig> is shown in figure 2b.
the fad-binding residues are located at the loop that connects strand β <dig> and helix α <dig>  therefore, it is reasonable to hypothesize that these hydrophobic residues would most likely participate in hydrophobic interactions to stabilize fad binding.
the etf:qo mutations of p.ala84thr and p.phe128ser were constructed in the same way as the wt structure.
both replacements are located at the center and peripheral of the hydrophobic patch, respectively .
molecular dynamics simulations
for the md simulations, the trajectories of the wt and mt etf:qo in the explicit solvent were calculated.
the backbone rmsd values for wt and mt etf:qo during the production phase relative to the starting structures were plotted .
these values were plotted to obtain an estimate of the md trajectory quality and convergence.
for the simulations of wt and mt etf:qo, the obtained data demonstrate that after a rapid increase during the first  <dig>  ns, the trajectories are stable with average values of  <dig> ,  <dig>  and  <dig>  Å for wt and the p.ala84thr and p.phe128ser etf:qo mutants, respectively.
statistical analysis of the rmsd data reveals that the trajectories are more stable after the first  <dig>  ns.
therefore, only the second half of the trajectory was analyzed.
based on zhang's study  <cit> , a segment corresponding to residues 71- <dig> of human etf:qo was found to form the fad-binding motif.
the binding site was located at the β1/α <dig> loop .
the rmsd of the fad-binding motif with respect to the starting conformation was compared for the wt and mt structures during the course of the simulations.
the rmsd was found to increase as a function of time for the mt p.ala84thr  and mt p.phe128ser  when compared with the wt.
the analysis of the b-factors for each residue at the β <dig>  α <dig>  α <dig>  and α <dig> regions revealed that the atomic fluctuations of the p.ala84thr and p.phe128ser mutants were significant at the β1/α <dig> loop  when compared with the wt values .
helix α <dig>  which may be involved in the support and stabilization of the α <dig> and α <dig> helices, was also found to have higher b-factors in both mutants .
moreover, the fluctuations of the mt p.phe128ser were also observed to be significant at helix α <dig> .
from the above mentioned analysis, it was observed that the mutations of p.ala84thr and p.phe128ser may induce additional fluctuations in the β1/α <dig> loop, α <dig>  and α <dig> regions.
such fluctuations may promote instability in this region of the protein and hamper fad binding.
normal mode analysis
the correlation map for etf:qo is shown in figure  <dig>  general structural elements of etf:qo can be identified by the pattern of the cross-correlations.
the enlargements along the diagonal are typical for α-helices, where motions of amino acids adjacent in the sequence are correlated, such as the elements of the helices α <dig>  α <dig> and α <dig> .
cross-correlations that extend as plumes from the diagonal correspond to anti-parallel elements of the secondary structure such as the strands β <dig>  β <dig>  β <dig> and β <dig> .
the cross-correlations are also found between the multiple structural elements, whereby some result from specific contacts, while others are longer range in nature.
for example, long-range cross-correlations are observed between the fad-binding motif  and helix α <dig> .
between the fad-binding motif and helix α <dig>  correlations exist because of the presence of a hydrophobic patch consisting of v <dig> , l <dig>  and f <dig>  .
helix α <dig> also displays significant correlated motion with the fad-binding motif and helix α <dig> .
these results suggest correlated dynamics between the fad-binding motif, helix α <dig> and helix α <dig>
discussion
in this study, we found that the p.ala84thr and p.phe128ser mutations are most likely to alter the protein structure near the fad binding site as well as disrupt the stability of the fad binding required for the activation of etf:qo using md simulations and nma.
interestingly, nma revealed that several reported disease-causing mutations in the etf:qo protein show highly correlated motions with the fad-binding site.
in the fad-binding family, the pyrophosphate moiety binds to the most strongly conserved sequence motif  <cit> .
a subset of the proteins that adopt the rossmann fold  <cit>  also bind to the nucleotide cofactors such as fad and nad, and function as oxidoreductases.
the rossmann fold can often be identified by the short amino-acid sequence motif, gxgxxg.
the pivotal role of the gly residues in the conserved central gxgxxg is well understood  <cit> .
the rossmann fold begins with a β-strand connected by a short loop to an α-helix  <cit> .
as previously described, the most conserved and well-studied sequence motif, xhxhgxgxxgxxxhxxh8hxhe, is part of the rossmann fold  <cit> .
here × is any residue and h is a hydrophobic residue.
the hydrophobic residues provide hydrophobic interactions between the α-helix and the β-sheet.
intriguingly, the mutation of p.ala84thr is located at a conserved hydrophobic residue of this motif of the rossmann fold .
in addition, the mutation of p.phe128ser is also located at this hydrophobic patch .
the above mentioned observations, along with the obtained results of the trajectory analyses , strongly suggest that these mutations could influence the structural stability of the fad-binding motif.
thus, it could lead to unstable fad binding.
therefore, we postulate that p.ala84thr and p.phe128ser may alter the stability of the fad-binding site and hamper efficient binding of the ligand.
in turn, this instability of the fad-binding site may impair the activity of the electron-transport system.
the binding of fad is important for the catalytic activity of flavoproteins as well as for the correct folding, assembly and protein stability  <cit> .
a reduced availability of intra-mitochondrial fad may result in a less stable or inactive conformation of the proteins.
as a result, an accelerated breakdown could result.
however, supplementation of riboflavin is most likely to increase the intra-mitochondrial fad concentration.
as a result, fad binding could be promoted.
this could ameliorate the effect of the mutations that reduce the affinity of etf:qo for fad  <cit> .
it should be noted that riboflavin treatment has been shown to ameliorate the symptoms and metabolic profiles in recent studies  <cit> .
if the charge or size of the amino acid is altered and the amino acid is buried within the core of the protein, folding may not proceed and the result is a complete loss of function.
such a loss of function has been found for a large number of variant short-chain acyl-coa dehydrogenase  and medium-chain acyl-coa dehydrogenase  proteins  <cit> .
in addition, it may also cause conformational changes to the 3d structure by altering its hydrophobicity, charge, overpacking, or cavity at the buried site, leading to a temperate damage  <cit> .
in the present study, we identified a novel mutation, p.phe128ser.
we have used a nma to characterize the detailed changes to protein fluctuations.
the results suggest that the dynamics of the fad-binding motif, helix α <dig> and helix α <dig> are correlated.
the substitution of p.phe128ser may alter the hydrophobic interactions between helix α <dig> and helix α <dig> .
it should be noted that numerous mutations near residue p.phe <dig> have been identified.
wen et al.
<cit>  recently identified three etfdh mutations, p.leu127arg, p.asp130val, and p.trp131cys in riboflavin-responsive madd patients in the north of china.
er et al.
<cit>  and liang et al.
<cit>  reported one etfdh mutation, p.leu127his, in riboflavin-responsive madd patients in the taiwanese population.
law et al.
<cit>  also reported one etfdh mutation, p.pro137ser, in a chinese family with riboflavin-responsive madd.
in addition, goodman et al.
<cit>  reported p.l138r as a disease-causing mutation.
interestingly, these mutations are located very close to each other in the region of the helix α <dig>  thus, these findings demonstrate the highly correlated motions of the fad-binding motif .
previously recognized etfdh mutations, p.arg175leu and p.arg175his  <cit> , were found in the region of helix α <dig>  both mutations have significantly correlated motions with the fad-binding motif, as shown in figure  <dig>  based on the present findings, we believe that the mutations to the amino acids in these regions are likely to affect the fad-binding stability.
therefore, it is highly recommended that the etfdh mutation in exon  <dig>   <dig> and  <dig> be initially screened.
however, based on the b-factors analysis, the atomic fluctuations of the p.leu127arg, and p.arg175his were not significant at the β1/α <dig> loop, which was involved in fad binding .
according to published literatures, these madd patients are compound heterozygous for two mutations.
therefore, it appears probable that other mutations most likely account for the disease state.
characterization of additional mutations in the other region of etf:qo will be established in future studies.
currently, the present study is the first to demonstrate through nma that several previously reported etfdh gene mutations have highly correlated motions with the fad-binding site.
CONCLUSIONS
the present study is the first demonstration of p.ala84thr and p.phe128ser by molecular modeling, md simulations, and correlated motion analysis.
along with the obtained results of the md simulations and nma, the present findings strongly suggest that p.ala84thr and p.phe128ser are likely to alter the etf:qo protein structure near the fad-binding site as well as disrupt the stability of fad binding, which is essential for the activation of etf:qo.
the change of hydrophobic interaction may destabilize fad binding, giving rise to a less stable or inactive conformation.
overall, the obtained findings have implications for the pathogenic mechanism of riboflavin-responsive madd.
competing interests
the authors declare that they have no competing interests.
authors' contributions
concept and design of the experiments: tke, ccc, yyl, hcc, jgc, jkh and yjj.
performance of the experiments: tke and ccc.
data analysis and discussion: tke, ccc, yyl, hcc, yhc, jgc, jkh and yjj.
contribution of reagents/materials/analysis tools: hcc, yhc, jgc and jkh.
manuscript preparation: tke, ccc, jgc, jkh and yjj.
all authors read and approved the final manuscript.
supplementary material
additional file 1
table s <dig>  dried blood spot acylcarnitine profile measured using tandem mass spectrometry in the patient before and after riboflavin treatment.
click here for file
additional file 2
table s <dig>  the highlighted amino acid alanine  and phenylalanine  shown in yellow and pink, respectively, at positions  <dig> and  <dig> are conserved in all the orthologs.
click here for file
additional file 3
figure s <dig>  rmsd plots of the whole wt and mt  p.a84t and  p.f128s structures with respect to the starting conformation during the course of the md simulations.
click here for file
additional file 4
figure s <dig>  structure of etf:qo with the positions of the identified amino acid changes near residue f <dig> in riboflavin-responsive madd patients labeled.
click here for file
additional file 5
figure s <dig>  structures of the wt and mt etf:qo are drawn in cartoon putty representation.
the red arrows indicate the positions of the mutations.
click here for file
acknowledgements
the present study was supported by a grant from the kaohsiung medical university hospital  and national science council .
we are grateful to both the hardware and software supports of the structural bioinformatics core facility at national chiao tung university.

 multiple acyl-coa dehydrogenase deficiency, also known as glutaric aciduria type ii, is an autosomal recessively inherited disorder of fatty acid metabolism. in the majority of cases, the disorder is caused by a defect in either the alpha- or beta-subunit of the electron transfer flavoprotein or the electron transfer flavoprotein dehydrogenase genes. however, in some patients, the disorder may result from a few, currently unidentified, disruptions to riboflavin metabolism. because of a deficiency of etf or etfdh, the acyl-coa dehydrogenases are unable to transfer the electrons generated by the dehydrogenation reactions. this results in the accumulation of various intramitochondrial acyl-coa esters. recently, etfdh mutations were reported to be major causes of riboflavin-responsive madd. characteristics such as organic aciduria may be detected only during periods of illness or catabolic stress. in the majority of cases, depending on the metabolic status of the patient, plasma acylcarnitine analysis may not provide a definitive diagnosis. amongst the most daunting challenges facing madd is metabolic heterogeneity, in particular, for the diagnosis of madd. thus, numerous molecular diagnostic methods have been established for analyzing the etf and/or etfdh genes for the purpose of achieving the final diagnosis of madd. previously, we reconfirmed the high prevalence of the c.250g > a mutation in taiwanese patients with riboflavin-responsive madd, as well as in the normal population using a high-resolution melting analysis. it should be noted that all the nine patients examined, including three pairs of siblings, harbor this mutation and four were homozygous. we estimated the allele frequency to be for the p.ala84thr mutant. thus, the estimated carrier frequency of this mutation in the taiwanese population is 1:. the homozygous mutant frequency was 1:. nevertheless, the carrier frequency of c.250g > a is estimated to be % amongst the population in southern china. thus, ~1: han chinese are expected to suffer from riboflavin-responsive madd. intriguingly, p.ala84thr has been identified in asian countries including taiwan, hong kong, singapore, thailand and southern china. a total of patients were examined from the above referenced literature and diagnosed with late-onset madd. amongst these patients, the mutation c.250g > a in the etfdh gene was detected in patients with homozygous mutations and patients with heterozygous mutations. to date, this mutation has never been reported in western countries. thus, it has been suggested that the most common mutation p.ala84thr is ethnic-specific. in addition, the mutation of p.ala84thr most likely accounts for the mild phenotype and response to riboflavin. here, a patient presented at 11/ years represented a mild to late-onset madd and the patient slightly improved following riboflavin treatment. the patient had compound heterozygous mutations of the etfdh gene. currently, there is no related study to explore the underlying mechanism for the most common mutation. flavin adenine dinucleotide is a cofactor for the electron transfer flavoprotein:ubiquinone oxidoreductase which constitutes the electron-transport pathway for a number of mitochondrial flavoprotein dehydrogenases that are mainly involved in fatty-acid and amino-acid metabolism. riboflavin treatment has been shown to strikingly ameliorate the symptoms and metabolic profiles in madd patients. this may enhance the conformational stabilization of the mutant etf:qo protein. two of the mutations are located in the fad-binding domain; however, the two amino acids do not have direct interactions with fad according to the predicted 3d structure of etf:qo. the closest distances between the mutations and the fad binding residues are and Å, respectively. therefore, to explore the effects of the mutations on etf:qo dynamics, molecular dynamics simulations of the wild type and mutant type etf:qo in the same model environment were compared. besides the md simulations, an alternative method, normal mode analysis, for studying protein motions was used to analyze the dynamic correlations between the mutation sites and the fad-binding motif. in this study, molecular modeling, md simulations and nma were performed to investigate the structural implications of the underlying mutations on the protein conformation. the obtained results may provide an insight into the pathomechanism of madd. methods patients the patient was a girl aged 11/ years who was clinically diagnosed with madd in her dried-blood spot sample was sent for acylcarnitine analysis. the results demonstrated elevation of acylcarnitines during the illness, consistent with glutaric aciduria type gas chromatography-mass spectrometry revealed significant elevations of urinary glutaric acid, 3-methylglutarate, 3-methylcluconate, adipate, 3-methyladipate, octenedioate, suberate, and hexanoylglycine. the present study was approved by the institutional review board of kaohsiung medical university hospital. genetic analysis the blood samples were collected from the patient and the family members as well as from normal controls with informed consents. the genomic dna samples were extracted from peripheral whole blood using the nucleospin® blood kit, according to the manufacturer's instructions. identification of mutations hrm analysis was applied for screening the mutation of etfdh, as previously described. pcr was carried out in duplicate in a μl final volume using the lightcycler ® high-resolution melting master × buffer which contained taq polymerase, nucleotides, the dye resolight and ng dna. the primers and mgcl were used at a concentration of μm and mm, respectively for identifying the etfdh gene mutations. the hrm assays were conducted using the lightcycler® instrument provided with the software lightcycler® gene-scanning software version. the pcr program required a sybr green i filter. the program consisted of an initial denaturation-activation step at 95°c for min, followed by a 45-cycle program. the 45-cycle program consisted of the following steps: denaturation at 95°c for s, annealing at 58°c or 60°c for s and elongation at 72°c for s with the reading of the fluorescence. the acquisition mode was single. the melting program consisted of three steps: denaturation at 95°c for min, renaturation at 40°c for min, and the subsequent melting that included continuous fluorescent readings from to 90°c at a rate of acquisitions per degree centigrade. to confirm the results of the hrm analysis, a sequencing analysis was also performed for all the samples. after the hrm analysis, the samples were purified using the favorprep™ pcr clean-up mini kit. the pcr products generated after hrm were sequenced directly. the sequence reaction was performed in a final volume of μl, comprising μl of the purified pcr product, μm of each pcr primer and μl of the abi prism terminator cycle sequencing kit v. the sequencing program started from 96°c for min and then followed by 25-cycle pcr program. the program consisted of the following steps: denaturation at 96°c for s; annealing at 50°c for s; and elongation at 60°c for min. the sequence detection was performed using the abi prism genetic analyzer. to ensure that the mutation found in the patient was the one specifically present in patients with madd, healthy control chromosomes were also screened. sorting intolerant from tolerant analysis sift tool generates multiple alignments of the sequences over different species to look at the conserved sequence of a gene; it asses the conserved amino acid positions and analyzes the effect of missense changes on the conserved structure of proteins over the course of evolution. the sift tool assigns a score to the mutations, and the score of < is considered potentially damaging. structure prediction of human wild type and mutant type etf:qo three-dimensional structures of the human etf:qo were predicted using the server. automatically uses an effective consensus strategy. the above mentioned strategy combines structural- and profile-based comparison methods for both template selection and target-template alignment. server selected the porcine etf:qo as the model template. the etf:qo models were built based on a sequence and structural alignment from the above mentioned template. a total of six models were yielded for each target by using a set of parameters. these models were based on the alternative target-template alignments. the qualities of the modeled structures were checked using ramachandran plots. the model with the highest quality was selected as the final predicted structure for the target. finally, the protein-ligand complex was constructed by superimposing the predicted human etf:qo model to the 3d structure of the porcine etf:qo-ligand complex. molecular dynamics simulations molecular dynamics simulations were performed using the software gromacs ver... opls-aa all-atom force field was used for the energy calculations. the models were solvated with simple point charge water molecules, and the models were simulated in a triclinic box with periodic boundary conditions. the simulations were performed in the canonical nvt ensemble. the energy of the models was first minimized using the steepest descent algorithm. this was followed by the position-restrained md simulations for ps. the linear constraint solver algorithm was used to constrain the bonds. the md simulations were carried out at constant pressure and temperature for ns using an integration time step of fs. the non-bonded interactions were cutoff at Å. at every time steps, the coordinates from the md simulations were saved. analysis of md trajectories the objective was to understand the structural and functional implications of the amino acid substitution. thus, the trajectories of wt and mt were analyzed for the following structural properties as a function of time: a) the root mean square deviation of the cα atoms with respect to the starting conformation; b) rmsd of the fad-binding motif with respect to the starting conformation; and c) the b-factors of the cα atoms which were calculated from the last ns of the md trajectories. the b-factors in protein structures reflect the fluctuation of atoms about their average positions. a large b-factor indicates high flexibility of individual atoms. normal mode analysis normal mode analysis is a powerful tool to estimate the dynamics based on structure. low-frequency normal modes describing the large-scale real-world motions of a protein have been demonstrated to be related to biological function. in the present study, the nma was performed using the software gromacs ver.. by diagonalization of the mass-weighted hessian matrix. the force field-based nma requires careful energy minimization of the initial structure. we first minimized the human etf:qo structure with the steepest descent integrator for a couple of steps. next, the l-bfgs integrator was used to minimize the maximum force close to zero and subsequently generate a hessian matrix. the nma was performed using the algorithm in gromacs. finally, the pair-wise residue correlation was calculated. the above mentioned correlation was calculated as the average correlation summed over lowest nonzero frequency normal modes.  patient and molecular diagnostic hrm analysis of etfdh gene revealed a novel t > c point mutation at position the point mutations were confirmed by direct dna sequencing. the hotspot mutation c.250g > a and a novel mutation c.383t > c in the etfdh gene were identified in this patient. these mutations are highly conserved amongst the different species. the sift tool analysis revealed a score of < and predicted that the replaced amino-acid was potentially damaging. this indicates that these mutations are most likely pathogenic. figures 1e and 1f show the melting profiles of the etfdh mutations in the patient's family. the patient had compound heterozygous mutations. the sibling was a carrier of c.383t > c. the father and mother were carriers of c.383t > c in exon and c.250g > a in exon respectively. the above mentioned mutation was not detected in the unrelated control chromosomes. the pedigree is presented in figure 1h. structure prediction of human etf:qo human etf:qo models were constructed using the published porcine etf:qo crystal structure as the template. a close-up view of the hydrophobic residues which are located at the fad-binding domain, including v i a v l v l and f is shown in figure 2b. the fad-binding residues are located at the loop that connects strand β and helix α therefore, it is reasonable to hypothesize that these hydrophobic residues would most likely participate in hydrophobic interactions to stabilize fad binding. the etf:qo mutations of p.ala84thr and p.phe128ser were constructed in the same way as the wt structure. both replacements are located at the center and peripheral of the hydrophobic patch, respectively. molecular dynamics simulations for the md simulations, the trajectories of the wt and mt etf:qo in the explicit solvent were calculated. the backbone rmsd values for wt and mt etf:qo during the production phase relative to the starting structures were plotted. these values were plotted to obtain an estimate of the md trajectory quality and convergence. for the simulations of wt and mt etf:qo, the obtained data demonstrate that after a rapid increase during the first ns, the trajectories are stable with average values of, and Å for wt and the p.ala84thr and p.phe128ser etf:qo mutants, respectively. statistical analysis of the rmsd data reveals that the trajectories are more stable after the first ns. therefore, only the second half of the trajectory was analyzed. based on zhang's study, a segment corresponding to residues 71- of human etf:qo was found to form the fad-binding motif. the binding site was located at the β1/α loop. the rmsd of the fad-binding motif with respect to the starting conformation was compared for the wt and mt structures during the course of the simulations. the rmsd was found to increase as a function of time for the mt p.ala84thr and mt p.phe128ser when compared with the wt. the analysis of the b-factors for each residue at the β α α and α regions revealed that the atomic fluctuations of the p.ala84thr and p.phe128ser mutants were significant at the β1/α loop when compared with the wt values. helix α which may be involved in the support and stabilization of the α and α helices, was also found to have higher b-factors in both mutants. moreover, the fluctuations of the mt p.phe128ser were also observed to be significant at helix α. from the above mentioned analysis, it was observed that the mutations of p.ala84thr and p.phe128ser may induce additional fluctuations in the β1/α loop, α and α regions. such fluctuations may promote instability in this region of the protein and hamper fad binding. normal mode analysis the correlation map for etf:qo is shown in figure general structural elements of etf:qo can be identified by the pattern of the cross-correlations. the enlargements along the diagonal are typical for α-helices, where motions of amino acids adjacent in the sequence are correlated, such as the elements of the helices α α and α. cross-correlations that extend as plumes from the diagonal correspond to anti-parallel elements of the secondary structure such as the strands β β β and β. the cross-correlations are also found between the multiple structural elements, whereby some result from specific contacts, while others are longer range in nature. for example, long-range cross-correlations are observed between the fad-binding motif and helix α. between the fad-binding motif and helix α correlations exist because of the presence of a hydrophobic patch consisting of v, l and f. helix α also displays significant correlated motion with the fad-binding motif and helix α. these results suggest correlated dynamics between the fad-binding motif, helix α and helix α discussion in this study, we found that the p.ala84thr and p.phe128ser mutations are most likely to alter the protein structure near the fad binding site as well as disrupt the stability of the fad binding required for the activation of etf:qo using md simulations and nma. interestingly, nma revealed that several reported disease-causing mutations in the etf:qo protein show highly correlated motions with the fad-binding site. in the fad-binding family, the pyrophosphate moiety binds to the most strongly conserved sequence motif. a subset of the proteins that adopt the rossmann fold also bind to the nucleotide cofactors such as fad and nad, and function as oxidoreductases. the rossmann fold can often be identified by the short amino-acid sequence motif, gxgxxg. the pivotal role of the gly residues in the conserved central gxgxxg is well understood. the rossmann fold begins with a β-strand connected by a short loop to an α-helix. as previously described, the most conserved and well-studied sequence motif, xhxhgxgxxgxxxhxxh8hxhe, is part of the rossmann fold. here × is any residue and h is a hydrophobic residue. the hydrophobic residues provide hydrophobic interactions between the α-helix and the β-sheet. intriguingly, the mutation of p.ala84thr is located at a conserved hydrophobic residue of this motif of the rossmann fold. in addition, the mutation of p.phe128ser is also located at this hydrophobic patch. the above mentioned observations, along with the obtained results of the trajectory analyses, strongly suggest that these mutations could influence the structural stability of the fad-binding motif. thus, it could lead to unstable fad binding. therefore, we postulate that p.ala84thr and p.phe128ser may alter the stability of the fad-binding site and hamper efficient binding of the ligand. in turn, this instability of the fad-binding site may impair the activity of the electron-transport system. the binding of fad is important for the catalytic activity of flavoproteins as well as for the correct folding, assembly and protein stability. a reduced availability of intra-mitochondrial fad may result in a less stable or inactive conformation of the proteins. as a result, an accelerated breakdown could result. however, supplementation of riboflavin is most likely to increase the intra-mitochondrial fad concentration. as a result, fad binding could be promoted. this could ameliorate the effect of the mutations that reduce the affinity of etf:qo for fad. it should be noted that riboflavin treatment has been shown to ameliorate the symptoms and metabolic profiles in recent studies. if the charge or size of the amino acid is altered and the amino acid is buried within the core of the protein, folding may not proceed and the result is a complete loss of function. such a loss of function has been found for a large number of variant short-chain acyl-coa dehydrogenase and medium-chain acyl-coa dehydrogenase proteins. in addition, it may also cause conformational changes to the 3d structure by altering its hydrophobicity, charge, overpacking, or cavity at the buried site, leading to a temperate damage. in the present study, we identified a novel mutation, p.phe128ser. we have used a nma to characterize the detailed changes to protein fluctuations. the results suggest that the dynamics of the fad-binding motif, helix α and helix α are correlated. the substitution of p.phe128ser may alter the hydrophobic interactions between helix α and helix α. it should be noted that numerous mutations near residue p.phe have been identified. wen et al. recently identified three etfdh mutations, p.leu127arg, p.asp130val, and p.trp131cys in riboflavin-responsive madd patients in the north of china. er et al. and liang et al. reported one etfdh mutation, p.leu127his, in riboflavin-responsive madd patients in the taiwanese population. law et al. also reported one etfdh mutation, p.pro137ser, in a chinese family with riboflavin-responsive madd. in addition, goodman et al. reported p.l138r as a disease-causing mutation. interestingly, these mutations are located very close to each other in the region of the helix α thus, these findings demonstrate the highly correlated motions of the fad-binding motif. previously recognized etfdh mutations, p.arg175leu and p.arg175his, were found in the region of helix α both mutations have significantly correlated motions with the fad-binding motif, as shown in figure based on the present findings, we believe that the mutations to the amino acids in these regions are likely to affect the fad-binding stability. therefore, it is highly recommended that the etfdh mutation in exon and be initially screened. however, based on the b-factors analysis, the atomic fluctuations of the p.leu127arg, and p.arg175his were not significant at the β1/α loop, which was involved in fad binding. according to published literatures, these madd patients are compound heterozygous for two mutations. therefore, it appears probable that other mutations most likely account for the disease state. characterization of additional mutations in the other region of etf:qo will be established in future studies. currently, the present study is the first to demonstrate through nma that several previously reported etfdh gene mutations have highly correlated motions with the fad-binding site.  the present study is the first demonstration of p.ala84thr and p.phe128ser by molecular modeling, md simulations, and correlated motion analysis. along with the obtained results of the md simulations and nma, the present findings strongly suggest that p.ala84thr and p.phe128ser are likely to alter the etf:qo protein structure near the fad-binding site as well as disrupt the stability of fad binding, which is essential for the activation of etf:qo. the change of hydrophobic interaction may destabilize fad binding, giving rise to a less stable or inactive conformation. overall, the obtained findings have implications for the pathogenic mechanism of riboflavin-responsive madd. competing interests the authors declare that they have no competing interests. authors' contributions concept and design of the experiments: tke, ccc, yyl, hcc, jgc, jkh and yjj. performance of the experiments: tke and ccc. data analysis and discussion: tke, ccc, yyl, hcc, yhc, jgc, jkh and yjj. contribution of reagents/materials/analysis tools: hcc, yhc, jgc and jkh. manuscript preparation: tke, ccc, jgc, jkh and yjj. all authors read and approved the final manuscript. supplementary material additional file 1 table s dried blood spot acylcarnitine profile measured using tandem mass spectrometry in the patient before and after riboflavin treatment. click here for file additional file 2 table s the highlighted amino acid alanine and phenylalanine shown in yellow and pink, respectively, at positions and are conserved in all the orthologs. click here for file additional file 3 figure s rmsd plots of the whole wt and mt p.a84t and p.f128s structures with respect to the starting conformation during the course of the md simulations. click here for file additional file 4 figure s structure of etf:qo with the positions of the identified amino acid changes near residue f in riboflavin-responsive madd patients labeled. click here for file additional file 5 figure s structures of the wt and mt etf:qo are drawn in cartoon putty representation. the red arrows indicate the positions of the mutations. click here for file acknowledgements the present study was supported by a grant from the kaohsiung medical university hospital and national science council. we are grateful to both the hardware and software supports of the structural bioinformatics core facility at national chiao tung university.
using molecular dynamics simulations and normal mode analysis, we found that the p.ala84thr and p.phe128ser mutations are most likely to alter the protein structure near the fad binding site as well as disrupt the stability of the fad binding required for the activation of etf:qo. recently, mutations in electron transfer flavoprotein dehydrogenase gene, encoding electron transfer flavoprotein:ubiquinone oxidoreductase have been reported to be the major causes of riboflavin-responsive madd. high resolution melting analysis and sequencing of the entire etfdh gene revealed a novel mutation and the hotspot mutation from a patient with madd. intriguingly, nma revealed that several reported disease-causing mutations in the etf:qo protein show highly correlated motions with the fad-binding site. based on the present findings, we conclude that the changes made to the amino acids in etf:qo are likely to influence the fad-binding stability. according to the predicted 3d structure of etf:qo, the two mutations are located within the flavin adenine dinucleotide binding domain; however, the two residues do not have direct interactions with the fad ligand.
 multiple acyl-coenzyme a dehydrogenase deficiency is an autosomal recessive disease caused by the defects in the mitochondrial electron transfer system and the metabolism of fatty acids. recently, mutations in electron transfer flavoprotein dehydrogenase gene, encoding electron transfer flavoprotein:ubiquinone oxidoreductase have been reported to be the major causes of riboflavin-responsive madd. to date, no studies have been performed to explore the functional impact of these mutations or their mechanism of disrupting enzyme activity.  high resolution melting analysis and sequencing of the entire etfdh gene revealed a novel mutation and the hotspot mutation from a patient with madd. according to the predicted 3d structure of etf:qo, the two mutations are located within the flavin adenine dinucleotide binding domain; however, the two residues do not have direct interactions with the fad ligand. using molecular dynamics simulations and normal mode analysis, we found that the p.ala84thr and p.phe128ser mutations are most likely to alter the protein structure near the fad binding site as well as disrupt the stability of the fad binding required for the activation of etf:qo. intriguingly, nma revealed that several reported disease-causing mutations in the etf:qo protein show highly correlated motions with the fad-binding site.  based on the present findings, we conclude that the changes made to the amino acids in etf:qo are likely to influence the fad-binding stability. 