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== Structure == | == Structure == | ||
[[Image:180429 proteopedia pura figures2.jpg|thumb|right| | [[Image:180429 proteopedia pura figures2.jpg|thumb|right|400px| A PUR domain is analogous to a left-handed handshake. PUR repeat I-II represented from [[5fgp]].]] | ||
Purα functions as a dimer composed of two intramolecular domains and one intermolecular domain. The Purα monomer contains three semi-conserved repeated amino acid sequences, named in order from N->C: PUR repeats I, II, and III. These repeats fold to form two domains: <scene name='78/786627/5fgp_intro/9'>PUR repeats I and II</scene> associating to form the I-II domain or “intramolecular domain”, while <scene name='78/786627/5fgo_repeatiii/3'>PUR repeat III</scene> facilitates dimerization through association with a repeat III from a second Purα monomer or repeat III of Purβ, forming an "intermolecular domain". Each PUR repeat is connected by a flexible linker region of 10-20 amino acids, depending on the species, linker and algorithm used to determine repeats. Each PUR repeat contains a beta-sheet composed of four beta-strands, followed by a single alpha-helix. While Purα is not yet officially classified by SCOP or CATH, its structure is that of an α+β protein. | Purα functions as a dimer composed of two intramolecular domains and one intermolecular domain. The Purα monomer contains three semi-conserved repeated amino acid sequences, named in order from N->C: PUR repeats I, II, and III. These repeats fold to form two domains: <scene name='78/786627/5fgp_intro/9'>PUR repeats I and II</scene> associating to form the I-II domain or “intramolecular domain”, while <scene name='78/786627/5fgo_repeatiii/3'>PUR repeat III</scene> facilitates dimerization through association with a repeat III from a second Purα monomer or repeat III of Purβ, forming an "intermolecular domain". Each PUR repeat is connected by a flexible linker region of 10-20 amino acids, depending on the species, linker and algorithm used to determine repeats. Each PUR repeat contains a beta-sheet composed of four beta-strands, followed by a single alpha-helix. While Purα is not yet officially classified by SCOP or CATH, its structure is that of an α+β protein. | ||
[[Image:180503 PurA Why2 | [[Image:180503 PurA Why2 comparison1.jpg|thumb|right|350px| PUR domains are structurally similar to the fold in Whirly proteins. Left: PUR repeat I-II ([[5fgp]]), right: WHY2 ([[3n1k]]).]] | ||
The domains of Purα have been described as "Whirly-like" folds because of their structural similarity to the DNA-binding Whirly class of proteins found in plants.<ref>PMID:19846792</ref> Whirlys are also ssDNA binding proteins, however unlike PUR family proteins they are not sequence-specific. | The domains of Purα have been described as "Whirly-like" folds because of their structural similarity to the DNA-binding Whirly class of proteins found in plants.<ref>PMID:19846792</ref> Whirlys are also ssDNA binding proteins, however unlike PUR family proteins they are not sequence-specific. | ||
The 2016 X-ray crystal structure of Purα repeat I-II ([[5fgp]]) shows DNA bound between repeats I and II. Residues indicated by this crystal structure to be involved in protein-DNA binding are: <scene name='78/786627/Repeati_resi/3'>(repeat I) Q52, S53 and K54</scene>; <scene name='78/786627/Repeatii_resi/1'>(repeat II) K138, N140, R142, and F145</scene>. K54 (repeat I) and K138 (repeat II) interact with the phosphate backbone, while Q52 (repeat I), S53 (repeat I), K54 (repeat I), R142 (repeat II), and N140 (repeat II) hydrogen bond directly with the DNA bases. Interestingly all bases appear to be stabilized by stacking interactions within the DNA strand except G4 which stacks directly with F145 in repeat II of the protein. This phenylalanine-guanine stacking interaction disrupts base-stacking within the DNA resulting in the neighboring cytosine being flipped out, introducing a strong kink in the DNA strand. This base-stacking interruption was shown to be responsible for Purα's DNA-unwinding activity by EMSA using a F145 mutant.<ref>PMID:26744780</ref> | The 2016 X-ray crystal structure of Purα repeat I-II ([[5fgp]]) shows DNA bound between repeats I and II. Residues indicated by this crystal structure to be involved in protein-DNA binding are: <scene name='78/786627/Repeati_resi/3'>(repeat I) Q52, S53 and K54</scene>; <scene name='78/786627/Repeatii_resi/1'>(repeat II) K138, N140, R142, and F145</scene>. K54 (repeat I) and K138 (repeat II) interact with the phosphate backbone, while Q52 (repeat I), S53 (repeat I), K54 (repeat I), R142 (repeat II), and N140 (repeat II) hydrogen bond directly with the DNA bases. Interestingly all bases appear to be stabilized by stacking interactions within the DNA strand except G4 which stacks directly with F145 in repeat II of the protein. This <scene name='78/786627/5fgp_57and145/3'>phenylalanine-guanine stacking interaction</scene> disrupts base-stacking within the DNA resulting in the neighboring cytosine being flipped out, introducing a strong kink in the DNA strand. This base-stacking interruption was shown to be responsible for Purα's DNA-unwinding activity by EMSA using a F145 mutant.<ref>PMID:26744780</ref> | ||
== Function == | == Function == | ||
Purα binds to single stranded purine-rich regions of DNA or RNA. In particular, Purα (and Purβ) are known to bind the purine-rich tract containing an MCAT enhancer motif in the 5’ region of the sense strand of the smooth muscle alpha actin gene (''Acta2''). Purα functions as a repressor at both transcriptional and translational levels. At the level of transcription Purα (and Purβ) act in concert with Y-box-binding protein 1 (YB-1 in humans or MSY1 in mice) which binds the antisense pyrimidine-rich strand, together acting to repress ''Acta2'' expression. At the level of translation Purα, Purβ and MSY-1 (YB-1 in humans) have been shown to bind a region in exon 3 of ''Acta2'' mRNA that is structurally similar to the MCAT (AGGAATG) enhancer element and prevent transcription in fibroblasts.<ref>PMID:10608902</ref> | Purα binds to single stranded purine-rich regions of DNA or RNA. In particular, Purα (and Purβ) are known to bind the purine-rich tract containing an MCAT enhancer motif in the 5’ region of the sense strand of the smooth muscle alpha actin gene (''Acta2''). Purα functions as a repressor at both transcriptional and translational levels. At the level of transcription Purα (and Purβ) act in concert with Y-box-binding protein 1 (YB-1 in humans or MSY1 in mice) which binds the antisense pyrimidine-rich strand, together acting to repress ''Acta2'' expression. At the level of translation Purα, Purβ and MSY-1 (YB-1 in humans) have been shown to bind a region in exon 3 of ''Acta2'' mRNA that is structurally similar to the MCAT (AGGAATG) enhancer element and prevent transcription in fibroblasts.<ref>PMID:10608902</ref> | ||
PurA has also been shown to enhance expression of myelin basic protein (MBP) in developing mouse brain tissue through its binding to an MB1 regulatory motif -15 to -40 nucleotides upstream of the transcription start site. MBP is an important protein found in myelin sheaths of the central nervous system.<ref>PMID:7657701</ref> | |||
[[Image:180504 PurA Repeat 1 electron density.jpg|thumb|right|350px|PUR repeat I electron density map showing base stacking between Y57 and guanine. The DNA strand present in this electron density map was omitted from the [[5fgp]] PDB file but is shown in the original publication (Weber, J. et al., 2016, eLIFE).<ref>PMID:26744780</ref> ([[5fgp]])]] | |||
High-affinity nucleic acid-binding function is dependent on Purα dimerization. Purα forms homodimers in addition to heterodimers with Purβ. PurA is known to repress various genes including SMαA. | High-affinity nucleic acid-binding function is dependent on Purα dimerization. Purα forms homodimers in addition to heterodimers with Purβ. PurA is known to repress various genes including SMαA. | ||
<scene name='78/786627/5fgp_57and145/1'>Two aromatic residues spatially conserved on PUR repeats I and II</scene>, Y57 (repeat I) and F145 (repeat II), located on the solvent-exposed surface of the beta-sheets, contribute to the DNA unwinding activity of Purα through base stacking interactions with DNA bases.<ref>PMID:26744780</ref> <scene name='78/786627/5fgo_repeatiii/4'>PUR repeat III also has an aromatic residue at this location (Y219)</scene> that could undergo base-stacking interactions with DNA, however Weber, et al. observed negligible unwinding activity in this repeat.<ref>PMID:26744780</ref> Furthermore, this group found repeat III to bind ssDNA with significantly less (~30-fold less) affinity than repeat I-II. | <scene name='78/786627/5fgp_57and145/1'>Two aromatic residues spatially conserved on PUR repeats I and II</scene>, Y57 (repeat I) and F145 (repeat II), located on the solvent-exposed surface of the beta-sheets, contribute to the DNA unwinding activity of Purα through base stacking interactions with DNA bases.<ref>PMID:26744780</ref> Although not shown in the [[5fgp]] PDB file, Y57 interacts with a second strand of DNA via base stacking interactions with guanine. It was presumably omitted because repeat I has significantly less affinity for purine-rich DNA than does repeat II, and it is believed that most of the DNA binding and unwinding activity occurs via interactions of DNA with the beta-sheet of repeat II.<ref>PMID:26744780</ref> <scene name='78/786627/5fgo_repeatiii/4'>PUR repeat III also has an aromatic residue at this location (Y219)</scene> that could undergo base-stacking interactions with DNA, however Weber, et al. observed negligible unwinding activity in this repeat.<ref>PMID:26744780</ref> Furthermore, this group found repeat III to bind ssDNA with significantly less (~30-fold less) affinity than repeat I-II. | ||
== Development == | == Development == | ||
Purα expression is highest during fetal development, | Purα expression is highest during fetal development, particularly in the brain and spinal cord. In humans and mice Purα expression is reduced in adulthood and, while still ubiquitous, levels are greatest in the brain. | ||
== Tissue Specificity == | == Tissue Specificity == | ||
| Line 31: | Line 33: | ||
*Purβ | *Purβ | ||
*Serum response factor (SRF) | *Serum response factor (SRF) | ||
*Y-box-binding protein 1 (YB-1) (MSY1 in mice) | *Sp1<ref>PMID:10457364</ref> | ||
*Retinoblastoma protein (Rb) | *E2F-1<ref>PMID:10597240</ref> | ||
*Rm62 RNA helicase | *Y-box-binding protein 1 (YB-1) (MSY1 in mice)<ref>PMID:10082537</ref> | ||
* | *Retinoblastoma protein (Rb)<ref>PMID:7592647</ref> | ||
*Rm62 RNA helicase (''Drosophila''), p68 RNA helicase (mammalian)<ref>PMID:21655086</ref> | |||
*JCV T-antigen<ref>PMID:8943069</ref> | |||
*Cyclin A/Cdk2 (PurA binds to the Cdk2 moiety)<ref>PMID:15707957</ref> | |||
== Disease == | == Disease == | ||
Mutations in the ''PURA'' gene resulting in haploinsufficiency are known to cause the neurological disease PURA syndrome, which has no cure. PURA syndrome appears early in development, with patients exhibiting severe developmental delay, seizures, feeding difficulty, intellectual disabilities, vision problems, hypotonia, and premature thelarche.<ref>PMID:29097605</ref> Purα knock-out mice exhibit similar neurological symptoms such as severe tremor developing at about postnatal week two, and feeding difficulties. These mice die after approximately one month. Heterozygous mice display less severe symptoms including seizures upon handling. <ref>PMID:25342064</ref> Interestingly, to date there have been no published accounts of phenotypic abnormalities in humans or mice with mutations in Purβ. | Mutations in the ''PURA'' gene resulting in haploinsufficiency are known to cause the neurological disease PURA syndrome, which has no cure. PURA syndrome appears early in development, with patients exhibiting severe developmental delay, seizures, feeding difficulty, intellectual disabilities, vision problems, hypotonia, and premature thelarche.<ref>PMID:29097605</ref> Purα knock-out mice exhibit similar neurological symptoms such as severe tremor developing at about postnatal week two, and feeding difficulties. These mice die after approximately one month. Heterozygous mice display less severe symptoms including seizures upon handling. <ref>PMID:25342064</ref> Interestingly, to date there have been no published accounts of phenotypic abnormalities in humans or mice with mutations in Purβ. | ||
Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is another disease associated with abnormal activity of Purα. In 2006, Johnson et al. observed that Purα colocalized with Fragile-X Mental Retardation Protein (FMRP) in mouse neuronal dendrites.<ref>PMID:16436378</ref> Purα also was found to be involved in dendritic mRNA transport, and in a ''Drosophila'' model of Fragile-X Tremor/Ataxia Syndrome it was hypothesized that the overexpression of FMR1 mRNA resulted in Purα being bound to these mRNAs leading to the apparent deficiency in Purα activity resulting in FXTAS symptoms.<ref>PMID:17698009</ref> | Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is another disease associated with abnormal activity of Purα. In 2006, Johnson et al. observed that Purα colocalized with Fragile-X Mental Retardation Protein (FMRP) in mouse neuronal dendrites.<ref>PMID:16436378</ref> Purα also was found to be involved in dendritic mRNA transport, and in a ''Drosophila'' model of Fragile-X Tremor/Ataxia Syndrome it was hypothesized that the overexpression of ''FMR1'' mRNA (encoding FMRP) resulted in Purα being bound to these mRNAs leading to the apparent deficiency in Purα activity and accumulation of RNA-protein inclusion bodies resulting in FXTAS symptoms.<ref>PMID:17698009</ref><ref>PMID:21655086</ref> | ||
== The Storybook Version of Pur Protein History (Just for Fun!) == | == The Storybook Version of Pur Protein History (Just for Fun!) == | ||