Structural Basis of Human Triosephosphate Isomerase Deficiency

Human triosephosphate isomerase deficiency is a rare autosomal disease that causes premature death of homozygous individuals. The most frequent mutation that leads to this illness is in position 104, which involves a conservative change of a Glu for Asp. Despite the extensive work that has been carried out on the E104D mutant enzyme in hemolysates and whole cells, the molecular basis of this disease is poorly understood. Here, we show that the purified, recombinant mutant enzyme E104D, while exhibiting normal catalytic activity, shows impairments in the formation of active dimers and low thermostability and monomerizes under conditions in which the wild type retains its dimeric form. The crystal structure of the E104D mutant at 1.85 Å resolution showed that its global structure was similar to that of the wild type; however, residue 104 is part of a conserved cluster of 10 residues, five from each subunit. An analysis of the available high resolution structures of TIM dimers revealed that this cluster forms a cavity that possesses an elaborate conserved network of buried water molecules that bridge the two subunits. In the E104D mutant, a disruption of contacts of the amino acid side chains in the conserved cluster leads to a perturbation of the water network in which the water-protein and water-water interactions that join the two monomers are significantly weakened and diminished. Thus, the disruption of this solvent system would stand as the underlying cause of the deficiency. Human triosephosphate isomerase deficiency is a rare autosomal disease that causes premature death of homozygous individuals. The most frequent mutation that leads to this illness is in position 104, which involves a conservative change of a Glu for Asp. Despite the extensive work that has been carried out on the E104D mutant enzyme in hemolysates and whole cells, the molecular basis of this disease is poorly understood. Here, we show that the purified, recombinant mutant enzyme E104D, while exhibiting normal catalytic activity, shows impairments in the formation of active dimers and low thermostability and monomerizes under conditions in which the wild type retains its dimeric form. The crystal structure of the E104D mutant at 1.85 Å resolution showed that its global structure was similar to that of the wild type; however, residue 104 is part of a conserved cluster of 10 residues, five from each subunit. An analysis of the available high resolution structures of TIM dimers revealed that this cluster forms a cavity that possesses an elaborate conserved network of buried water molecules that bridge the two subunits. In the E104D mutant, a disruption of contacts of the amino acid side chains in the conserved cluster leads to a perturbation of the water network in which the water-protein and water-water interactions that join the two monomers are significantly weakened and diminished. Thus, the disruption of this solvent system would stand as the underlying cause of the deficiency. Triosephosphate isomerase (TIM 3The abbreviations used are:TIM or TPItriosephosphate isomeraseHsTIMhuman TIMMOPS3-(N-morpholino)propanesulfonic acid. or TPI), a ubiquitous, essential glycolytic enzyme, catalyzes the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (1Albery W.J. Knowles J.R. Biochemistry. 1976; 15: 5631-5640Crossref PubMed Scopus (592) Google Scholar, 2Knowles J.R. Nature. 1991; 350: 121-124Crossref PubMed Scopus (505) Google Scholar). The functional structure of all known TIMs is assembled from monomers of about 250 residues into homodimers or tetramers. The crystal structure of the enzyme from 17 different species has been described, including the human enzyme (HsTIM) (3Mande S.C. Mainfroid V. Kalk K.H. Goraj K. Martial J.A. Hol W.G. Protein Sci. 1994; 3: 810-821Crossref PubMed Scopus (124) Google Scholar, 4Kinoshita T. Maruki R. Warizaya M. Nakajima H. Nishimura S. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2005; 61: 346-349Crossref PubMed Scopus (28) Google Scholar). Despite the fact that each monomer has a full set of catalytic residues, the enzyme is only active in its oligomeric state (5Waley S.G. Biochem. J. 1973; 135: 165-172Crossref PubMed Scopus (91) Google Scholar, 6Zabori S. Rudolph R. Jaenicke R. Z. Naturforsch. C. 1980; 35: 999-1004Crossref PubMed Scopus (80) Google Scholar). triosephosphate isomerase human TIM 3-(N-morpholino)propanesulfonic acid. In humans, TIM deficiencies are a rare class of autosomal diseases that are clinically manifested by chronic hemolytic anemia, neuromuscular disorders, neurological alterations, and cardiomyopathy and that result in the death of affected individuals within the first 5 years of age. The deficiencies are due to mutations in the TIM gene, with the most frequent occurring in the codon for amino acid 104 that leads to the replacement of Glu by Asp (reviewed in Ref. 7Schneider A.S. Baillieres Best Pract. Res. Clin. Haematol. 2000; 13: 119-140Crossref PubMed Scopus (103) Google Scholar). In studies of hemolysates from patients affected by TIM deficiency, the activity of the enzyme is found to be more than an order of magnitude lower than in normal individuals. Further, this reduction is accompanied by accumulation of dihydroxyacetone phosphate to levels several times higher than those of the controls (7Schneider A.S. Baillieres Best Pract. Res. Clin. Haematol. 2000; 13: 119-140Crossref PubMed Scopus (103) Google Scholar, 8Ationu A. Humphries A. Lalloz M.R. Arya R. Wild B. Warrilow J. Morgan J. Bellingham A.J. Layton D.M. Blood. 1999; 94: 3193-3198Crossref PubMed Google Scholar, 9Orosz F. Vertessy B.G. Hollan S. Horanyi M. Ovadi J. J. Theor. Biol. 1996; 182: 437-447Crossref PubMed Scopus (24) Google Scholar, 10Ovadi J. Orosz F. Hollan S. Mol. Cell Biochem. 2004; 256: 83-93Crossref PubMed Google Scholar, 11Orosz F. Olah J. Alvarez M. Keseru G.M. Szabo B. Wagner G. Kovari Z. Horanyi M. Baroti K. Martial J.A. Hollan S. Ovadi J. Blood. 2001; 98: 3106-3112Crossref PubMed Scopus (28) Google Scholar, 12Repiso A. Boren J. Ortega F. Pujades A. Centelles J. Vives-Corrons J.L. Climent F. Cascante M. Carreras J. Haematologica. 2002; 87: ECR12PubMed Google Scholar, 13Olah J. Orosz F. Puskas L.G. Hackler Jr., L. Horanyi M. Polgar L. Hollan S. Ovadi J. Biochem. J. 2005; 392: 675-683Crossref PubMed Scopus (38) Google Scholar, 14Hollan S. Magocsi M. Fodor E. Horanyi M. Harsanyi V. Farkas T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10362-10366Crossref PubMed Scopus (24) Google Scholar). At high dihydroxyacetone phosphate S. J. Biol. 2005; PubMed Scopus (28) Google this be to the of TIM In mutations in TIM to the accumulation of S. R. Horanyi M. Baroti K. Hollan S. PubMed Scopus (124) Google Scholar, B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The of normal and mutant to and has been F. Olah J. Alvarez M. Keseru G.M. Szabo B. Wagner G. Kovari Z. Horanyi M. Baroti K. Martial J.A. Hollan S. Ovadi J. Blood. 2001; 98: 3106-3112Crossref PubMed Scopus (28) Google Scholar, F. Wagner G. K. J. Baroti K. Horanyi M. Farkas T. Hollan S. Ovadi J. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). In was that of the E104D and is higher than in the The low TIM activity in patients was at in to this higher on the of E104D been in from in from patients that the or in with the mutant F. Vertessy B.G. Hollan S. Horanyi M. Ovadi J. J. Theor. Biol. 1996; 182: 437-447Crossref PubMed Scopus (24) Google Scholar, 10Ovadi J. Orosz F. Hollan S. Mol. Cell Biochem. 2004; 256: 83-93Crossref PubMed Google Scholar, 11Orosz F. Olah J. Alvarez M. Keseru G.M. Szabo B. Wagner G. Kovari Z. Horanyi M. Baroti K. Martial J.A. Hollan S. Ovadi J. Blood. 2001; 98: 3106-3112Crossref PubMed Scopus (28) Google Scholar, 12Repiso A. Boren J. Ortega F. Pujades A. Centelles J. Vives-Corrons J.L. Climent F. Cascante M. Carreras J. Haematologica. 2002; 87: ECR12PubMed Google Scholar, R. J. Biol. PubMed Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The are with TIM activity of affected individuals lower than in was that TIM activity in the E104D mutant is A. Boren J. Ortega F. Pujades A. Centelles J. Vives-Corrons J.L. Climent F. Cascante M. Carreras J. Haematologica. 2002; 87: ECR12PubMed Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). system in was used to the between the two monomers of mutant including E104D M. G. M. H. S. PubMed Scopus Google Scholar). In all was that is a weakened between the two Despite extensive work that has been carried out on the E104D mutant enzyme in hemolysates and whole cells, the of the enzyme been the of the to as to of the enzyme or the in or whole cells, we the and of recombinant E104D TIM and with those of the recombinant wild type The that the of the mutant and wild type are of the by and revealed that the enzyme is In the showed that the wild type enzyme and the mutant enzyme different wild type has two the mutant has only of the and of the wild type and recombinant from with show that the mutant dimers are than the wild type dimers and that the formation of active dimers from monomers is lower in the mutant than in the wild In to the structure of the mutant enzyme, is that the crystal structure of and human TIM at and Å used to the low activity that been in patients affected by this which is in the of the enzyme, at Å of the active was that a of in the would lower the of the with an perturbation of the active (3Mande S.C. Mainfroid V. Kalk K.H. Goraj K. Martial J.A. Hol W.G. Protein Sci. 1994; 3: 810-821Crossref PubMed Scopus (124) Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). we that the crystal structure of the E104D mutant would more the crystal structure of E104D at 1.85 Å resolution revealed that the of the catalytic residues was and that the most of the E104D mutation is the disruption of a conserved water network that the and to be essential for the of TIM and of Wild Human and E104D the of wild type was by Martial The was into the was in order to a on the amino of the enzyme on a a In a was on the as in Ref. Mol. Biol. 2002; 182: Google The mutant E104D was on this the The into at in and an of was At that for with The was used to the E104D The of from a was in of phosphate and 10 by and at for The was on a 10 of The was with a of and for a and The was recombinant from the Protein Sci. 1999; PubMed Scopus Google and to The was at a of of of and at for The was on and A. 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Sect. 1994; PubMed Scopus Google Scholar). and structure was by the molecular replacement with the A.J. J. Crystallogr. PubMed Scopus Google the of the at Å resolution T. Maruki R. Warizaya M. Nakajima H. Nishimura S. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2005; 61: 346-349Crossref PubMed Scopus (28) Google as the was with the G.M. J. M. T. Acta Crystallogr. Sect. PubMed Scopus Google by with K. Acta Crystallogr. Sect. Biol. Crystallogr. 2004; PubMed Scopus Google Scholar). The of the E104D mutation was by the structure of the wild type At the used for the that the monomers of the The the of the of water all residues and or are on the are poorly in all In of the monomers is more than the due to of the that the and active of the used to the molecules first in and a was on a and on a the to at between and used to the of the and the of water and are in with on the and of the in are for the resolution of of of molecules in of from in used on the used on the in a the and The of water molecules was Acta Crystallogr. Sect. 1994; PubMed Scopus Google Scholar). molecules with an of than 10 as and of conserved water molecules the K. K. Res. PubMed Scopus Google Scholar). water molecules with the V. J. K. J. Crystallogr. Google Scholar). of the was the E. K. J. Mol. Biol. PubMed Scopus Google Scholar). and with the J. Z. J. A. J. Res. PubMed Scopus Google Scholar). The E104D the as the Wild order to the low TIM activity that is in hemolysates of patients that the E104D mutation is due to an low activity of the mutant enzyme, we the of wild type and its E104D In the of glyceraldehyde 3-phosphate to dihydroxyacetone the for the and for the wild type and the mutant enzyme, the and for the wild type and the E104D mutant, The two The of the and the of from in the E104D than in Wild has been that in and in from patients affected by the E104D TIM activity is A. Boren J. Ortega F. Pujades A. Centelles J. Vives-Corrons J.L. Climent F. Cascante M. Carreras J. Haematologica. 2002; 87: ECR12PubMed Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google we the of the wild type and mutant at The of the mutant enzyme at or about a in activity that was higher than in the wild The of activity at high was significantly in the mutant enzyme than in the wild type; for at the E104D mutant most of its activity in in the the wild type about of its activity The at of the wild type and the E104D mutant as of The E104D mutant a with an at The wild type a different In the of a that a of structure was as the of the was was a of structure with an of is that the of the mutant and wild type are similar The of wild type and the E104D mutant by The mutant a with a of In the of the wild type two with of and in at the wild type retains about of the activity, which the of a dimeric state of the enzyme at this Thus, by the of the mutant enzyme is lower than that of wild which is in with the found at the was the two of the wild type and that of the mutant at lower that are all under Thus, the mutation E104D causes a in the on the showed that only a of those in the first that the wild type and E104D mutant the of the E104D mutant we the mutant and the wild type at different for at at that the of the was TIM monomers are (5Waley S.G. Biochem. J. 1973; 135: 165-172Crossref PubMed Scopus (91) Google Scholar, 6Zabori S. Rudolph R. Jaenicke R. Z. Naturforsch. C. 1980; 35: 999-1004Crossref PubMed Scopus (80) Google Scholar, V. G. H. E. G. R. M. A. Biochemistry. PubMed Scopus Google was that at the between would be a in the activity of the was in the wild type and the mutant however, at in which the activity of the mutant enzyme was the wild type The lower of the dimers of the E104D mutant that the mutation the of we the formation of active dimers from monomers found that in the mutant and the wild type the of as the of in the was at all of the of the of the wild type was higher than that of the E104D in the E104D mutant, the of was the in the of is that in this enzyme, the of the monomers is The that in with the wild the between monomers and dimers in the mutant enzyme is the this by of the wild type and mutant enzyme at The with the mutant showed that as the of the that to the this was accompanied by the of a with the molecular of the monomer In the the wild type only the that to the a that to was The of the E104D of the with the Wild the molecular basis of the of the E104D mutant its crystal structure was at 1.85 Å molecular replacement and an a showed that the mutation was in the monomers of the and The of as by of by the low in the of the mutant enzyme with the wild type in the of the active or the between the wild type and the E104D be the on the crystal structures of and human that the mutation interactions in the of the mutation that to perturbation of the catalytic at the of and (3Mande S.C. Mainfroid V. Kalk K.H. Goraj K. Martial J.A. Hol W.G. Protein Sci. 1994; 3: 810-821Crossref PubMed Scopus (124) Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Here, we found or of 104 in the of a of analysis of the TIM structures from different species showed that residue 104 is part of a conserved cluster of residues by and between the two and The cluster is in all of the TIMs to in the which are and in which of residues are conserved H. B. R. J. Mol. Biol. 2001; PubMed Scopus Google Scholar, M. A. S. H. M.R. Acta Crystallogr. Sect. Biol. Crystallogr. PubMed Scopus Google Scholar). that is essential for monomer be essential for dimeric and In the the two are to with a of about 10 Å between residues 104 of the two subunits. in the five residues of each of the two in the wild type are by a set of however, the only side contacts are by of and of the In the acid cluster is part of the cavity of the enzyme, with an of and a of In each of the TIM structures described, this cavity is with water of the in 5 is that the interactions between the amino the cluster are the interactions that are by with amino from are The analysis of water molecules in TIMs from different species at resolution of Å or including the Å resolution of TIM from shows a that with the cluster of conserved residues and In the whole conserved water molecules of which are in the of the active of monomer The an solvent of or than 10 that are buried and are all in the of residue 104 and for to wild type the of all of the water molecules is which that is an water in each of the two is that all of the conserved water molecules in the of the be however, as and of water molecules are conserved in the crystal structures of from and at and Å or from at Å and A. which are times active than dimeric part of the known as which the residues of the cluster that the conserved water network is the of the two The E104D the of the and the structures of E104D and wild type in the interactions between the residues and water molecules that the conserved cluster and In to in the mutant TIM contacts with and In the wild two with in the E104D mutant, is only the of a of the of the between and and in to the wild type the residue was the an in the of to of the that the between the two residues is than in the wild type between the residues that are in with residue 104 The contacts that are at Å are in and the contacts in more than Å from the wild type enzyme are in wild and in a of the water network in wild type and the E104D TIM showed In the wild and this water is in the mutant The contacts of and with residue 104 in the wild type are in the mutant enzyme and in the wild type enzyme, the water molecules The of the interactions with a in the of and The of the of conserved water molecules from all TIMs showed that of has the of all of the water the in the E104D mutant, the are higher than the In in the mutant enzyme, the contacts of the water with and are 5 and the of the conserved water with and with on the mutant enzyme contacts with and and In the wild type enzyme, and are at the to with residues and the and of contacts in the E104D mutant with the solvent molecules to the water network in the cluster a than that of the wild type the of conserved and water molecules on the the of a of interactions that the two is a in all is in the E104D the show that at of the of the conserved cluster of residues is to a water network that is in the E104D mutation a of side contacts that the conserved network of water In this we the recombinant E104D mutant of human TIM in order to a conservative mutation that at the of the in and death of affected individuals. the known human TIM the E104D is the only in is that the mutant E104D or a is in all affected individuals with TIM deficiency, homozygous and (reviewed in Ref. 7Schneider A.S. Baillieres Best Pract. Res. Clin. Haematol. 2000; 13: 119-140Crossref PubMed Scopus (103) Google Scholar). that hemolysates of patients with the E104D mutation low activity F. Vertessy B.G. Hollan S. Horanyi M. Ovadi J. J. Theor. Biol. 1996; 182: 437-447Crossref PubMed Scopus (24) Google Scholar, 10Ovadi J. Orosz F. Hollan S. Mol. Cell Biochem. 2004; 256: 83-93Crossref PubMed Google Scholar, 11Orosz F. Olah J. Alvarez M. Keseru G.M. Szabo B. Wagner G. Kovari Z. Horanyi M. Baroti K. Martial J.A. Hollan S. Ovadi J. Blood. 2001; 98: 3106-3112Crossref PubMed Scopus (28) Google Scholar, 12Repiso A. Boren J. Ortega F. Pujades A. Centelles J. Vives-Corrons J.L. Climent F. Cascante M. Carreras J. Haematologica. 2002; 87: ECR12PubMed Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). we found that the and the of the wild type and the mutant Thus, would that the low TIM activity in patients with the E104D mutation is due to of its catalytic that for the low activity in patients that the In of the of the mutant is its low The show that at high the mutant enzyme activity than the wild which is in with the in hemolysates A. Boren J. Ortega F. Pujades A. Centelles J. Vives-Corrons J.L. Climent F. Cascante M. Carreras J. Haematologica. 2002; 87: ECR12PubMed Google and from patients affected by the E104D mutation R. J. Biol. PubMed Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). this and showed that of the wild type at about higher than in the mutant In this is that studies on the of the at showed that the dimers that the E104D mutation than the wild type is that the of the mutant monomers is lower than that of the wild in with the of M. G. M. H. S. PubMed Scopus Google that show that the of the mutant monomers is lower than in the wild Thus, the to the wild in the E104D mutant, the between monomers and dimers is the was found by that the mutant enzyme was under conditions that about of activity, was an in the of monomers to dimers in the enzyme in the wild In this result in the accumulation of In is that the low activity in TIM deficiency patients is to a of and dimeric mutant species due to lower In order to a more at the molecular into the impairments of the E104D mutant we the crystal structure of the The crystal structures of all of the TIMs show that the residues of which into the for the of the contacts between the two subunits. is that of the residues that only and are conserved in all of the available TIM that are in the of In this we to to the cluster by five residues of each of the two of this cluster is with the of the residues that this are conserved in all of the that is that of the residues from each monomer that part of the with the E. K. J. Mol. Biol. PubMed Scopus Google this is the only with and The cluster forms a cavity that is with water of which are conserved in all of the crystal structures of TIM of the are buried low with the of the solvent molecules that are on all the TIM on each of more than contacts with an amino acid residue or water The high of of amino acid residues and water molecules in the cluster in all dimeric with the on the low of E104D mutant that the of this of the is to the and of the In the the only between the two is a bridge and The of this is by the of Mainfroid V. M. S.C. Hol W.G. Martial J.A. Goraj K. J. Mol. Biol. 1996; PubMed Scopus Google which show that the mutation in an the of the contacts that with residues are water In the E104D mutant enzyme, the bridge is the mutation about an in the between and that in a from the crystal structures of the wild type and mutant would that the of the mutations in this would be to the conserved network of water molecules that the two of the in the mutant E104D, we the of a conserved water to residue 104 on the wild an of the of two conserved water molecules that are to residue 104 on and the of several and that are in the wild In we show a conservative change on the of an leads to in the formation and of a by the crystal structure of the E104D mutant and by a analysis of the TIM structures from different was found that solvent molecules a in the of TIM In all the disruption of the water network is the underlying cause of the and of the E104D TIM deficiency. has been that mutations that are to or part of the of be with diseases T. R. K. J. Mol. Biol. PubMed Scopus Google Scholar, J.L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J.A. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). is known that the of an extensive and buried solvent is a of structure (reviewed in F. J. 2005; PubMed Scopus Google Scholar, Struct. 35: PubMed Google Scholar, Struct. Biol. PubMed Scopus Google to this is the first that a between a perturbation of and a disease has been in the crystal structure of the enzyme at Å resolution The enzyme was and under conditions similar to those for the mutant The analysis of this structure the in the at the at the of the was by the of of of under The of and is The of and is with