Mutations in HOXD13 Underlie Syndactyly Type V and a Novel Brachydactyly-Syndactyly Syndrome
2007; Elsevier BV; Volume: 80; Issue: 2 Linguagem: Inglês
10.1086/511387
ISSN1537-6605
AutoresXiuli Zhao, Miao Sun, Jin Zhao, J. Alfonso Leyva, Hongwen Zhu, Wei Yang, Xuan Zeng, Yang Ao, Qing Liu, Guoyang Liu, Wilson H.Y. Lo, Ethylin Wang Jabs, L. Mario Amzel, Xiangnian Shan, Xue Zhang,
Tópico(s)Developmental Biology and Gene Regulation
ResumoHOXD13, the homeobox-containing gene located at the most 5′ end of the HOXD cluster, plays a critical role in limb development. It has been shown that mutations in human HOXD13 can give rise to limb malformations, with variable expressivity and a wide spectrum of clinical manifestations. Polyalanine expansions in HOXD13 cause synpolydactyly, whereas amino acid substitutions in the homeodomain are associated with brachydactyly types D and E. We describe two large Han Chinese families with different limb malformations, one with syndactyly type V and the other with limb features overlapping brachydactyly types A4, D, and E and mild syndactyly of toes 2 and 3. Two-point linkage analysis showed LOD scores >3 (θ=0) for markers within and/or flanking the HOXD13 locus in both families. In the family with syndactyly type V, we identified a missense mutation in the HOXD13 homeodomain, c.950A→G (p.Q317R), which leads to substitution of the highly conserved glutamine that is important for DNA-binding specificity and affinity. In the family with complex brachydactyly and syndactyly, we detected a deletion of 21 bp in the imperfect GCN (where N denotes A, C, G, or T) triplet-containing exon 1 of HOXD13, which results in a polyalanine contraction of seven residues. Moreover, we found that the mutant HOXD13 with the p.Q317R substitution was unable to transactivate the human EPHA7 promoter. Molecular modeling data supported these experimental results. The calculated interactions energies were in agreement with the measured changes of the activity. Our data established the link between HOXD13 and two additional limb phenotypes—syndactyly type V and brachydactyly type A4—and demonstrated that a polyalanine contraction in HOXD13, most likely, led to other digital anomalies but not to synpolydactyly. We suggest the term "HOXD13 limb morphopathies" for the spectrum of limb disorders caused by HOXD13 mutations. HOXD13, the homeobox-containing gene located at the most 5′ end of the HOXD cluster, plays a critical role in limb development. It has been shown that mutations in human HOXD13 can give rise to limb malformations, with variable expressivity and a wide spectrum of clinical manifestations. Polyalanine expansions in HOXD13 cause synpolydactyly, whereas amino acid substitutions in the homeodomain are associated with brachydactyly types D and E. We describe two large Han Chinese families with different limb malformations, one with syndactyly type V and the other with limb features overlapping brachydactyly types A4, D, and E and mild syndactyly of toes 2 and 3. Two-point linkage analysis showed LOD scores >3 (θ=0) for markers within and/or flanking the HOXD13 locus in both families. In the family with syndactyly type V, we identified a missense mutation in the HOXD13 homeodomain, c.950A→G (p.Q317R), which leads to substitution of the highly conserved glutamine that is important for DNA-binding specificity and affinity. In the family with complex brachydactyly and syndactyly, we detected a deletion of 21 bp in the imperfect GCN (where N denotes A, C, G, or T) triplet-containing exon 1 of HOXD13, which results in a polyalanine contraction of seven residues. Moreover, we found that the mutant HOXD13 with the p.Q317R substitution was unable to transactivate the human EPHA7 promoter. Molecular modeling data supported these experimental results. The calculated interactions energies were in agreement with the measured changes of the activity. Our data established the link between HOXD13 and two additional limb phenotypes—syndactyly type V and brachydactyly type A4—and demonstrated that a polyalanine contraction in HOXD13, most likely, led to other digital anomalies but not to synpolydactyly. We suggest the term "HOXD13 limb morphopathies" for the spectrum of limb disorders caused by HOXD13 mutations. The homeobox-containing (HOX) genes are a highly conserved transcription-factor family that displays important function in early development.1Krumlauf R Hox genes in vertebrate development.Cell. 1994; 78: 191-201Abstract Full Text PDF PubMed Scopus (1690) Google Scholar, 2Capecchi MR Hox genes and mammalian development.Cold Spring Harb Symp Quant Biol. 1997; 62: 273-281Crossref PubMed Google Scholar In humans, there are 39 HOX genes arranged in four separate clusters: HOXA, HOXB, HOXC, and HOXD.3Scott MP A rational nomenclature for vertebrate homeobox (HOX) genes.Nucleic Acids Res. 1993; 21: 1687-1688Crossref PubMed Scopus (71) Google Scholar These clusters are located on chromosomes 7p15, 17q21, 12q13, and 2q31, respectively, and show a striking colinearity in their 5′→3′ genomic position and transcription direction. HOXD13 (MIM *142989; GenBank accession number NM_000523), the most 5′ gene of the HOXD cluster, has two coding exons: exon 1 with the imperfect GCN (where N denotes A, C, G, or T) triplet repeats encoding the N-terminal region with a 15-residue polyalanine tract (residues 49–63), and exon 2, which contains the homeobox region encoding the C-terminal portion with a 60-residue homeodomain (residues 268–327).4Akarsu AN Stoilov I Yilmaz E Sayli BS Sarfarazi M Genomic structure of HOXD13 gene: a nine polyalanine duplication causes synpolydactyly in two unrelated families.Hum Mol Genet. 1996; 5: 945-952Crossref PubMed Scopus (130) Google Scholar The homeodomain forms three α-helices. 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Synpolydactyly (SPD [MIM 186000], or syndactyly type II) is a dominantly inherited limb malformation with incomplete penetrance and variable expressivity. It is characterized by soft-tissue syndactyly between fingers 3 and 4 and between toes 4 and 5, with partial or complete digit duplication within the syndactylous web. Both interfamilial and intrafamilial variations in SPD limb phenotype exist. Individuals with an atypical form of SPD share a distinctive set of foot phenotypes, including small spurs of bone between metatarsals 1 and 2.11Goodman FR Limb malformations and the human HOX genes.Am J Med Genet. 2002; 112: 256-265Crossref PubMed Scopus (176) Google Scholar Polyalanine-expansion mutations in HOXD13 lead to typical SPD, whereas deletions and missense mutations are associated with atypical SPD.11Goodman FR Limb malformations and the human HOX genes.Am J Med Genet. 2002; 112: 256-265Crossref PubMed Scopus (176) Google Scholar, 12Muragaki Y Mundlos S Upton J Olsen BR Altered growth and branching patterns in synpolydactyly caused by mutations in HOXD13.Science. 1996; 272: 548-551Crossref PubMed Scopus (468) Google Scholar, 13Goodman FR Mundlos S Muragaki Y Donnai D Giovannucci-Uzielli ML Lapi E Majewski F McGaughran J McKeown C Reardon W et al.Synpolydactyly phenotypes correlate with size of expansions in HOXD13 polyalanine tract.Proc Natl Acad Sci USA. 1997; 94: 7458-7463Crossref PubMed Scopus (177) Google Scholar, 14Baffico M Baldi M Cassan PD Costa M Mantero R Garani P Camera G Synpolydactyly: clinical and molecular studies on four Italian families.Eur J Hum Genet Suppl. 1997; 5: 142Google Scholar, 15Kjaer KW Hedeboe J Bugge M Hansen C Friis-Henriksen K Vestergaard MB Tommerup N Opitz JM HOXD13 polyalanine tract expansion in classical synpolydactyly type Vordingborg.Am J Med Genet. 2002; 110: 116-121Crossref PubMed Scopus (22) Google Scholar, 16Kjaer KW Hansen L Eiberg H Utkus A Skovgaard LT Leicht P Opitz JM Tommerup N A 72-year-old Danish puzzle resolved—comparative analysis of phenotypes in families with different-sized HOXD13 polyalanine expansion.Am J Med Genet A. 2005; 138: 328-339Crossref PubMed Scopus (16) Google Scholar, 17Zhao XL Meng JP Sun M Ao Y Wu AH Lo HY Zhang X HOXD13 polyalanine tract expansion in synpolydactyly: mutation detection and prenatal diagnosis in a large Chinese family.Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2005; 22: 5-9PubMed Google Scholar, 18Goodman FR Giovannucci-Uzielli ML Hall C Reardon W Winter R Scambler P Deletions in HOXD13 segregate with an identical, novel foot malformation in two unrelated families.Am J Hum Genet. 1998; 63: 992-1000Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 19Calabrese O Bigoni S Gualandi F Trabanelli C Camera G Calzolari E A new mutation in HOXD13 associated with foot pre-postaxial polydactyly.Eur J Hum Genet Suppl. 2000; 8: 140Google Scholar, 20Kan SH Johnson D Giele H Wilkie AO An acceptor splice site mutation in HOXD13 results in variable hand, but consistent foot malformations.Am J Med Genet A. 2003; 121: 69-74Crossref Scopus (30) Google Scholar, 21Debeer P Bacchelli C Scambler PJ De Smet L Fryns JP Goodman FR Severe digital abnormalities in a patient heterozygous for both a novel missense mutation in HOXD13 and a polyalanine tract expansion in HOXA13.J Med Genet. 2002; 39: 852-856Crossref PubMed Google Scholar Furthermore, in the spontaneous spdh mouse mutant, which has a phenotype similar to human SPD, a polyalanine expansion of seven residues in Hoxd13 was detected.22Johnson KR Sweet HO Donahue LR Ward-Bailey P Bronson RT Davisson MT A new spontaneous mouse mutation of Hoxd13 with a polyalanine expansion and phenotype similar to human synpolydactyly.Hum Mol Genet. 1998; 7: 1033-1038Crossref PubMed Scopus (80) Google Scholar, 23Albrecht AN Schwabe GC Stricker S Boddrich A Wanker EE Mundlos S The synpolydactyly homolog (spdh) mutation in the mouse—a defect in patterning and growth of limb cartilage elements.Mech Dev. 2002; 112: 53-67Crossref PubMed Scopus (42) Google Scholar Missense mutations in HOXD13 also underlie brachydactyly types D (BDD [MIM 113200]) and E (BDE [MIM 113300]).24Johnson D Kan S-h Oldridge M Trembath RC Roche P Esnouf RM Giele H Wilkie AOM Missense mutations in the homeodomain of HOXD13 are associated with brachydactyly types D and E.Am J Hum Genet. 2003; 72: 984-997Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 25Caronia G Goodman FR McKeown CME Scambler PJ Zappavigna V An I47L substitution in the HOXD13 homeodomain causes a novel human limb malformation by producing a selective loss of function.Development. 2003; 130: 1701-1712Crossref PubMed Scopus (61) Google Scholar BDD presents with short and broad distal phalanges of the thumbs and halluces, whereas BDE has the cardinal feature of one or more shortened metacarpals and/or metatarsals. Two HOXD13 missense mutations within the homeodomain—p.S308C and p.I314L—have been described in families exhibiting features of BDE with BDD and BDE with mild SPD, respectively.24Johnson D Kan S-h Oldridge M Trembath RC Roche P Esnouf RM Giele H Wilkie AOM Missense mutations in the homeodomain of HOXD13 are associated with brachydactyly types D and E.Am J Hum Genet. 2003; 72: 984-997Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 25Caronia G Goodman FR McKeown CME Scambler PJ Zappavigna V An I47L substitution in the HOXD13 homeodomain causes a novel human limb malformation by producing a selective loss of function.Development. 2003; 130: 1701-1712Crossref PubMed Scopus (61) Google Scholar Syndactyly type V (MIM 186300), defined as "syndactyly associated with metacarpal and metatarsal synostosis" by Temtamy and McKusick,26Temtamy SA McKusick VA Syndactyly.in: Bergsma T The genetics of hand malformations. Alan R. Liss, New York1978: 301-322Google Scholar(p302) represents one of the rarest types of nonsyndromic syndactyly.27Malik S Ahmad W Grzeschik KH Koch MC A simple method for characterizing syndactyly in clinical practice.Genet Couns. 2005; 16: 229-238PubMed Google Scholar It is inherited as an autosomal dominant trait with synostotic fusion of metacarpals 4 and 5 as the hallmark.28Robinow M Johnson GF Brook GJ Syndactyly type V.Am J Med Genet. 1982; 11: 475-482Crossref PubMed Scopus (16) Google Scholar Brachydactyly type A4 (BDA4 [MIM 112800]) is characterized by short middle phalanges of the 2nd and 5th fingers and absence of middle phalanges of the 2nd–5th toes. In this report, we describe limb phenotypes of syndactyly type V and a novel brachydactyly-syndactyly syndrome that includes BDA4, and we report novel mutations in HOXD13 in two large Han Chinese families. Clinical findings.—We investigated two large Han Chinese families with distinctive limb malformations. Both families had affected females and male-to-male transmission (fig. 1A and 1B), consistent with autosomal dominant inheritance. There were 23 affected individuals in the 6 generations of family 1 (fig. 1A). At the proband's request, 13 affected individuals were physically examined, and digital photographs were taken. Among those individuals, 11 had hand and foot radiographs. On radiological examination, nine affected individuals had bilateral or unilateral fusion of metacarpals 4 and 5 (fig. 2B, 2F, 2J, and 2N). Radiographs showed that the fusion was variable in extent. The proband had a complete fusion in his right hand (fig. 2B), and eight other affected individuals had bilateral complete fusion (fig. 2F, 2J, and 2N). In five affected individuals, the fusion extended to the phalanges of fingers 4 and 5 (fig. 2N). Other hand deformities included ulnar deviation of fingers 2–5 (12 of 13 subjects), lobster claw–like or Y-shaped fingers 4 and 5 with an angulated 5th finger (8 of 13), shortening or clinodactyly of the 5th fingers (3 of 13), short distal phalanges of between one and all fingers (12 of 13), shortening of fused metacarpals 4 and 5 or metacarpal 5 (6 of 13), unilateral cutaneous syndactyly of fingers 3 and 4 (4 of 13), interdigital clefts between fingers 3 and 4 (10 of 13), camptodactyly (10 of 13), and absence of distal interphalangeal creases (13 of 13) (fig. 2A, 2B, 2E, 2F, 2I, 2J, 2M, and 2N). None of the affected individuals had metatarsal fusion of the feet (fig. 2D, 2H, 2L, and 2P). The most constant foot deformities were varus deviation of the first metatarsals; valgus deviation of toes 1–4; hyperplasia of the 1st ray, affecting the first metatarsals and the phalanges of the halluces; hypoplasia and shortness of metatarsals 2–5; and shortened and tucked 5th toes (fig. 2C, 2D, 2G, 2H, 2K, 2L, 2O, and 2P). Mild cutaneous syndactyly of toes 2 and 3 or 3 and 4 was observed in four affected individuals (IV-5, IV-8, IV-10, and V-13) (fig. 2K). Of note, one affected individual (V-9) with bilateral complete fusion of metacarpals 4 and 5 also had postaxial polydactyly in his left hand, as well as hypospadias. Taken together, the phenotypes in this family closely resemble the syndactyly type V reported by Robinow and colleagues.28Robinow M Johnson GF Brook GJ Syndactyly type V.Am J Med Genet. 1982; 11: 475-482Crossref PubMed Scopus (16) Google ScholarFigure 2.Photographs and radiographs of the proband (A–D), individual V-10 (E–H), individual V-13 (I–L), and individual IV-10 (M–P) of family 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Family 2 also had 23 affected individuals in 6 generations (fig. 1B). All 17 patients who are still alive were available for phenotype evaluation. Digital photographs and radiographs were taken for 16 and 13 of them, respectively. On clinical examination, most of the patients exhibited generalized shortening of hands and feet, 11 displayed broad and short distal phalanges of the thumbs (fig. 3A, 3E, 3I, and 3M), and 14 had mild cutaneous syndactyly of toes 2 and 3 (fig. 3C, 3G, 3K, and 3O). Radiographs revealed a constant phenotypic feature in all 13 patients: absence of middle phalanges of toes 2–5 (fig. 3D, 3H, 3L, and 3P) and very marked short middle phalanges of the 5th finger (fig. 3B, 3F, 3J, and 3N). Combined shortening of the middle phalanges was noticed, with patterns 2–5 (3 patients), 2-4-5 (3 patients), and 2-3-4-5 (2 patients), which indicates that the 2nd and 5th fingers are most severely affected (fig. 3B, 3F, 3J, and 3N). In many cases, the shortened middle phalanges were fused with the distal ones (fig. 3B, 3F, 3J, and 3N). In 12 patients, radiographs also showed very pronounced shortening of metacarpal 5, either alone or in combination with metatarsal 5 and/or other metacarpals/metatarsals (fig. 3B, 3D, 3F, 3H, 3J, 3L, 3N, and 3P). Short proximal phalanges of toes 1, 3, and 4 were apparent in 7 patients (fig. 3H, 3L, and 3P). Other common limb anomalies included broad first metatarsals and hallux phalanges, often associated with hallux valgus (fig. 3C, 3D, 3G, 3H, 3K, 3L, 3O, and 3P). Notably, the proband also had small spurs of bone between the 1st and 2nd metatarsals (fig. 3D). Almost all limb anomalies in this family were bilateral. These limb phenotypes overlap with those of BDA4, BDD, and BDE, and syndactyly type I (MIM %185900). No family member showed short stature or polydactyly. Molecular genetics.—We performed two-point linkage analysis and mutation identification after obtaining informed consent from participating family members and approval of Peking Union Medical College Institutional Review Board. Blood samples were collected and genomic DNA was extracted from 27 members of family 1, including 13 patients, and from 43 family members of family 2, including 15 patients. Six polymorphic microsatellite markers from chromosome region 2q24.3-q32.1, including a CA-repeat sequence within the intron of the HOXD13 gene, were selected and typed in the families, for two-point linkage analysis. LOD scores were calculated using the MLINK program of the LINKAGE package. The parameters used in linkage analysis were autosomal dominant inheritance, complete penetrance, a mutation rate of zero, equal male-female recombination rate, equal microsatellite-allele frequency, and a disease-allele frequency of 1 in 10,000. Maximal LOD scores of 4.90 in family 1 and of 6.46 in family 2 were obtained for marker D2S2314 at θ=0.00, showing definitive evidence of linkage. Haplotype analysis indicated no recombination at five genetic markers (D2S2981, D2S2314, D13-CA, D2S324, and D2S1391) in family 1 (fig. 1A) and 3 markers (D2S2981, D2S2314, and D13-CA) in family 2 (fig. 1B), indicating that the disease locus was linked to the chromosome region harboring HOXD13. We searched for pathogenic mutations in the proband of family 1 by direct sequencing of the PCR-amplified DNA fragments spanning exons 1 and 2 of HOXD13. We identified a missense mutation, c.950A→G (p.Q317R), substituting an arginine (R) for the highly conserved Q50, which is important for DNA-binding specificity and affinity (fig. 4A). To confirm this missense mutation, we introduced into the 950G mutant allele a BamHI recognition sequence (5′-GGATCC-3′), using a mismatch primer (5′-CTTGTCCTTCACTCTTCGGATC-3′) in a semi-nested PCR. Restriction analysis with use of this BamHI site in all available members from family 1 and in 136 unrelated control individuals of similar ethnic background revealed the presence of the mutation in all affected individuals but not in unaffected family members and control individuals (fig. 4A). In the proband of family 2, we sequenced the entire HOXD13 gene, including a promoter region of 1.5 kb, two exons, one intron, two UTRs (5′-UTR and 3′-UTR), and the coding exons of HOXD9 (GenBank accession number NM_014213), HOXD10 (GenBank accession number NM_002148), HOXD11 (GenBank accession number NM_021192), and HOXD12 (GenBank accession number NM_021193). We found a deletion of 21 bp in the imperfect GCN triplet–repeat sequence of HOXD13 exon 1, c.157_177del, which results in a polyalanine contraction of seven residues (p.A53_A59del) (fig. 4B). By polyacrylamide gel electrophoresis, it was confirmed that this deletion cosegregated with the limb phenotypes in affected individuals but was not detected in any unaffected individuals of the family or in 500 unrelated Han Chinese control individuals. A 4-alanine contraction was detected in the control individuals, with an allele frequency of 0.3% (3 in 1,000). We also identified a novel missense mutation in HOXD11—c.734G→A (p.G245D)—on the same chromosome that carries the 7-alanine contraction mutation in HOXD13. This HOXD11 mutation was detected in 2 of 139 unrelated Han Chinese control individuals. Functional study.—We conducted the luciferase reporter assay to determine the consequences of the p.Q317R substitution in transactivating the promoter of EPHA7 (GenBank accession number NM_004440), one of the direct downstream target genes of HOXD13 during limb development.29Salsi V Zappavigna V Hoxd13 and Hoxa13 directly control the expression of the EphA7 ephrin tyrosine kinase receptor in developing limbs.J Biol Chem. 2006; 281: 1992-1999Crossref PubMed Scopus (48) Google Scholar The p.I314L and p.Q317R mutations in HOXD13 change the amino acids at the 47th and 50th residues of the homeodomain, respectively. "I47L" had been used elsewhere to designate the p.I314L mutation.25Caronia G Goodman FR McKeown CME Scambler PJ Zappavigna V An I47L substitution in the HOXD13 homeodomain causes a novel human limb malformation by producing a selective loss of function.Development. 2003; 130: 1701-1712Crossref PubMed Scopus (61) Google Scholar Similarly, we named the mutant HOXD13 with the p.Q317R substitution "Q50R." The human EPHA7 promoter of 660 bp (from −580 to +80), which contains an HOXD13-binding site ATATTATGG, was obtained by PCR from human genomic DNA through use of primers EPHA7BamF 5′-CGCGGATCCTGTTCGCTCGCACCGT-3′ and EPHA7R 5′-AGACTTCCTTTCCCACTCCC-3′. The amplified fragment was cloned into the pGL3-basic vector (Promega) at the BglII and KpnI sites upstream of the firefly luciferase gene and was verified by sequencing (fig. 5A). To make the expression vectors for FLAG-HOXD13WT (the FLAG-tagged wild type) and FLAG-HOXD13−7A (the FLAG-tagged mutant with the 7-alanine contraction), the full coding regions were generated by genomic PCR from normal and affected individuals, respectively. The verified fragments were then cloned into the HindIII and SmaI sites of the p3×FLAG-CMV-7 plasmid (Sigma), to produce the pFLAG-HOXD13WT and pFLAG-HOXD13−7A expression vectors, respectively. pFLAG-HOXD13Q50R and pFLAG-HOXD13I47L were constructed from the pFLAG-HOXD13WT by site-directed mutagenesis with use of the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The primer pairs used were 5′-CCATTTGGTTTCGGAACCGAAGAGTG-3′ and 5′-CACTCTTCGGTTCCGAAACCAAATGG-3′ for Q50R and 5′-GACAAGTGACCCTTTGGTTTCAG-3′ and 5′-CTGAAACCAAAGGGTCACTTGTC-3′ for I47L. The reporter construct (1.0 μg) was cotransfected with 1.5 μg of one of the four test constructs (pFLAG-HOXD13WT, pFLAG-HOXD13Q50R, pFLAG-HOXD13I47L, or pFLAG-HOXD13−7A) into the C3H10T1/2 mouse embryonic fibroblast cells growing in a 12-well plate, with use of 1.0 μl VigoFect reagent (Vigorous Biotechnology). The pRL-SV40 Renilla luciferase vector (25 ng) was also added in each cotransfection, to normalize the transfection efficiency. Cells were lysed and assayed for luciferase activity following the Dual Luciferase protocol (Promega). Consistent with the recent findings that the mouse EphA7 promoter could mediate transcriptional activation by HOXD13, expression of the human wild-type HOXD13WT could also transactivate the human EPHA7 promoter (fig. 5B).29Salsi V Zappavigna V Hoxd13 and Hoxa13 directly control the expression of the EphA7 ephrin tyrosine kinase receptor in developing limbs.J Biol Chem. 2006; 281: 1992-1999Crossref PubMed Scopus (48) Google Scholar However, a remarkable difference in the decrease of transactivation between the HOXD13Q50R and HOXD13I47L mutants was observed (fig. 5B and 5C). Q50R more severely impaired HOXD13′s capacity to transactivate the human EPHA7 promoter, retaining only 13% of the reporter activity compared with the wild-type counterpart, whereas I47L showed merely moderate impairment, with 63% of reporter activity remaining (fig. 5C). The polyalanine contraction of seven residues seemed to exert no significant effect on HOXD13-induced transactivation (fig. 5B and 5C). Cotransfection of pFLAG-HOXD13WT and pFLAG-HOXD13−7A showed no significant change in transcriptional activity (data not shown). Molecular modeling.—We modeled the interaction between the Q50R-mutant HOXD13 and DNA on the basis of the crystal structure of Drosophila melanogaster antenna pedia (antp) homeodomain (Protein Data Bank ID 9ANT) bound to DNA (fig. 6A). The Antp/DNA complex (PDB ID: 9ANT) was used as template for the modeling experiments.30Fraenkel E Pabo CO Comparison of X-ray and NMR structures for the Antennapedia homeodomain-DNA complex.Nature Struct Biol. 1998; 5: 692-697PubMed Google Scholar I47 and Q50 of the homeodomain are identical in 9ANT and HOXD13. The base corresponding to the 3′-thymidine (Thy) in the core DNA consensus sequence 5′-TAAT-3′ is Thy221 in 9ANT. To study the role of residues 47 and 50 of the HOXD13 homeodomain and the interacting base (Thy), four models in the protein-DNA interface were generated in silico (with the programs QUANTA [Accelrys] and Program O31Kleywegt GJ Jones TA Efficient rebuilding of protein structures.Acta Crystallogr D Biol Crystallogr. 1996; 52: 829-832Crossref PubMed Scopus (163) Google Scholar) from the crystal structure, to mimic the different interactions. Two amino acids were mutated in the homeodomain: I47→leucine (L) and Q50→arginine (R). The backbone conformation was not altered. The molecules were placed in a box containing 6,400 water molecules. All calculations were performed with NAMD32Kalé L Skeel R Bhandarkar M Brunner R Gursoy A Krawetz N Phillips J Shinozaki A Varadarajan K Schulten K NAMD2: greater scalability for parallel molecular dynamics.J Comput Phys. 1999; 151: 283-312Crossref Scopus (2051) Google Scholar with use of the CHARMM33MacKerell Jr, AD Bashford D Bellott M Dunbrack Jr, RL Evanseck JD Field MJ Fischer S Gao J Guo H Ha S et al.All-atom empirical potential for molecular modeling and dynamics studies of proteins.J Phys Chem B. 1998; 102: 3586-3616Crossref Scopus (10704) Google Scholar force field. To calculate the electrostatic interactions, a cutoff of 0.20 nm was used. The energy of the structures was minimized by 75,000 steps by use of the conjugated gradient and line-search algorithm of NAMD. Previous NMR and molecular dynamics studies have shown the dynamic, fluctuating nature of the protein-DNA interactions.34Qian YQ Otting G Wüthrich K NMR1 detection of hydration water in the intermolecular interface of a protein-DNA complex.J Am Chem Soc. 1993; 115: 1189-1190Crossref Scopus (83) Google Scholar, 35Billeter M Guntert P Luginbuhl P Wüthrich K Hydration and DNA recognition by homeodomains.Cell. 1996; 85: 1057-1065Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar Therefore, the complete homeodomain and the DNA were allowed to move during the minimization, to relax the entire structure and to ensure more-reliable calculations. To study the local influence of mutations, only the interaction energies between the pairs Q/R (I/L) and Thy were calculated with NAMD and the CHARMM force field, respectively. Drawings were prepared with PyMOL (DeLano Scientific) and VMD.36Humphrey W Dalke A Schulten K VMD: visual molecular dynamics.J Mol Graph. 1996; 14: 33-38Crossref PubMed Scopus (31531) Google Scholar In an attempt to estimate the differences among the models, the interaction energy was calculated for each of the combinations of amino acid and base. The difference in the energies for the Q50R mutation was not as small as in the I47L mutation (fig. 6B). The van der Waals interactions were of the same order for both mutations, which suggests that the change in the steric hindrance was not significant. The electrostatic contribution to the interaction
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