scholarly journals Structural basis for the recognition of the scaffold protein Frmpd4/Preso1 by the TPR domain of the adaptor protein LGN

2015 ◽  
Vol 71 (2) ◽  
pp. 175-183 ◽  
Author(s):  
Hiroki Takayanagi ◽  
Satoru Yuzawa ◽  
Hideki Sumimoto
Keyword(s):  
Crystal Structure ◽  
Consensus Sequence ◽  
Adaptor Protein ◽  
Structural Basis ◽  
Tetratricopeptide Repeat ◽  
Amino Acid Residues ◽  
Binding Region ◽  
Α Helix ◽  
Tpr Domain ◽  
Partner Proteins

The adaptor protein LGN interactsviathe N-terminal domain comprising eight tetratricopeptide-repeat (TPR) motifs with its partner proteins mInsc, NuMA, Frmpd1 and Frmpd4 in a mutually exclusive manner. Here, the crystal structure of the LGN TPR domain in complex with human Frmpd4 is described at 1.5 Å resolution. In the complex, the LGN-binding region of Frmpd4 (amino-acid residues 990–1011) adopts an extended structure that runs antiparallel to LGN along the concave surface of the superhelix formed by the TPR motifs. Comparison with the previously determined structures of the LGN–Frmpd1, LGN–mInsc and LGN–NuMA complexes reveals that these partner proteins interact with LGN TPR1–6viaa common core binding region with consensus sequence (E/Q)XEX4–5(E/D/Q)X1–2(K/R)X0–1(V/I). In contrast to Frmpd1, Frmpd4 makes additional contacts with LGNviaregions N- and C-terminal to the core sequence. The N-terminal extension is replaced by a specific α-helix in mInsc, which drastically increases the direct contacts with LGN TPR7/8, consistent with the higher affinity of mInsc for LGN. A crystal structure of Frmpd4-bound LGN in an oxidized form is also reported, although oxidation does not appear to strongly affect the interaction with Frmpd4.

scholarly journals Tah1 helix-swap dimerization prevents mixed Hsp90 co-chaperone complexes

2015 ◽  
Vol 71 (5) ◽  
pp. 1197-1206 ◽  
Author(s):  
Rhodri M. L. Morgan ◽  
Mohinder Pal ◽  
S. Mark Roe ◽  
Laurence H. Pearl ◽  
Chrisostomos Prodromou
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Tetratricopeptide Repeat ◽  
Normal Complement ◽  
Methionine Residue ◽  
Client Proteins ◽  
Α Helix ◽  
Tpr Domain

Specific co-chaperone adaptors facilitate the recruitment of client proteins to the Hsp90 system. Tah1 binds the C-terminal conserved MEEVD motif of Hsp90, thus linking an eclectic set of client proteins to the R2TP complex for their assembly and regulation by Hsp90. Rather than the normal complement of seven α-helices seen in other tetratricopeptide repeat (TPR) domains, Tah1 unusually consists of the first five only. Consequently, the methionine of the MEEVD peptide remains exposed to solvent when bound by Tah1. In solution Tah1 appears to be predominantly monomeric, and recent structures have failed to explain how Tah1 appears to prevent the formation of mixed TPR domain-containing complexes such as Cpr6–(Hsp90)2–Tah1. To understand this further, the crystal structure of Tah1 in complex with the MEEVD peptide of Hsp90 was determined, which shows a helix swap involving the fifth α-helix between two adjacently bound Tah1 molecules. Dimerization of Tah1 restores the normal binding environment of the bound Hsp90 methionine residue by reconstituting a TPR binding site similar to that in seven-helix-containing TPR domain proteins. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes.

Crystal structure of the complex of the interaction domains of Escherichia coli DnaB helicase and DnaC helicase loader: structural basis implying a distortion-accumulation mechanism for the DnaB ring opening caused by DnaC binding

2019 ◽  
Vol 167 (1) ◽  
pp. 1-14
Author(s):  
Koji Nagata ◽  
Akitoshi Okada ◽  
Jun Ohtsuka ◽  
Takatoshi Ohkuri ◽  
Yusuke Akama ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Molecular Model ◽  
Structural Basis ◽  
Terminal Domain ◽  
Helicase Dnab ◽  
Accumulation Mechanism ◽  
Interaction Domains ◽  
Α Helix ◽  
Available Information

Abstract Loading the bacterial replicative helicase DnaB onto DNA requires a specific loader protein, DnaC/DnaI, which creates the loading-competent state by opening the DnaB hexameric ring. To understand the molecular mechanism by which DnaC/DnaI opens the DnaB ring, we solved 3.1-Å co-crystal structure of the interaction domains of Escherichia coli DnaB–DnaC. The structure reveals that one N-terminal domain (NTD) of DnaC interacts with both the linker helix of a DnaB molecule and the C-terminal domain (CTD) of the adjacent DnaB molecule by forming a three α-helix bundle, which fixes the relative orientation of the two adjacent DnaB CTDs. The importance of the intermolecular interface in the crystal structure was supported by the mutational data of DnaB and DnaC. Based on the crystal structure and other available information on DnaB–DnaC structures, we constructed a molecular model of the hexameric DnaB CTDs bound by six DnaC NTDs. This model suggested that the binding of a DnaC would cause a distortion in the hexameric ring of DnaB. This distortion of the DnaB ring might accumulate by the binding of up to six DnaC molecules, resulting in the DnaB ring to open.

scholarly journals The crystal structure of human forkhead box N1 in complex with DNA reveals the structural basis for forkhead box family specificity

2019 ◽  
Vol 295 (10) ◽  
pp. 2948-2958 ◽  
Author(s):  
Joseph A. Newman ◽  
Hazel Aitkenhead ◽  
Angeline E. Gavard ◽  
Ioanna A. Rota ◽  
Adam E. Handel ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Consensus Sequence ◽  
Family Members ◽  
Structural Basis ◽  
Thymic Epithelial Cell ◽  
Thymic Involution ◽  
Forkhead Box ◽  
Recognition Helix ◽  
Dna Specificity ◽  
Α Helix

Forkhead box N1 (FOXN1) is a member of the forkhead box family of transcription factors and plays an important role in thymic epithelial cell differentiation and development. FOXN1 mutations in humans and mice give rise to the “nude” phenotype, which is marked by athymia. FOXN1 belongs to a subset of the FOX family that recognizes an alternative forkhead-like (FHL) consensus sequence (GACGC) that is different from the more widely recognized forkhead (FKH) sequence RYAAAYA (where R is purine, and Y is pyrimidine). Here, we present the FOXN1 structure in complex with DNA containing an FHL motif at 1.6 Å resolution, in which the DNA sequence is recognized by a mixture of direct and water-mediated contacts provided by residues in an α-helix inserted in the DNA major groove (the recognition helix). Comparisons with the structure of other FOX family members revealed that the FKH and FHL DNA sequences are bound in two distinct modes, with partially different registers for the protein DNA contacts. We identified a single alternative rotamer within the recognition helix itself as an important determinant of DNA specificity and found protein sequence features in the recognition helix that could be used to predict the specificity of other FOX family members. Finally, we demonstrate that the C-terminal region of FOXN1 is required for high-affinity DNA binding and that FOXN1 has a significantly reduced affinity for DNA that contains 5′-methylcytosine, which may have implications for the role of FOXN1 in thymic involution.

scholarly journals Crystal structure of the Schizosaccharomyces pombe U7BR E2-binding region in complex with Ubc7

2019 ◽  
Vol 75 (8) ◽  
pp. 552-560
Author(s):  
Zachary S. Hann ◽  
Meredith B. Metzger ◽  
Allan M. Weissman ◽  
Christopher D. Lima
Keyword(s):  
Crystal Structure ◽  
Saccharomyces Cerevisiae ◽  
Quality Control ◽  
Schizosaccharomyces Pombe ◽  
Protein Quality ◽  
Back Side ◽  
Binding Region ◽  
Sequence Alignments ◽  
Structural Differences ◽  
Α Helix

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a protein quality-control pathway in eukaryotes in which misfolded ER proteins are polyubiquitylated, extracted and ultimately degraded by the proteasome. This process involves ER membrane-embedded ubiquitin E2 and E3 enzymes, as well as a soluble E2 enzyme (Ubc7 in Saccharomyces cerevisiae and UBE2G2 in mammals). E2-binding regions (E2BRs) that recruit these soluble ERAD E2s to the ER have been identified in humans and S. cerevisiae, and structures of E2–E2BR complexes from both species have been determined. In addition to sequence and structural differences between the human and S. cerevisiae E2BRs, the binding of E2BRs also elicits different biochemical outcomes with respect to E2 charging by E1 and E2 discharge. Here, the Schizosaccharomyces pombe E2BR was identified and purified with Ubc7 to resolve a 1.7 Å resolution co-crystal structure of the E2BR in complex with Ubc7. The S. pombe E2BR binds to the back side of the E2 as an α-helix and, while differences exist, it exhibits greater similarity to the human E2BR. Structure-based sequence alignments reveal differences and conserved elements among these species. Structural comparisons and biochemistry reveal that the S. pombe E2BR presents a steric impediment to E1 binding and inhibits E1-mediated charging, respectively.

scholarly journals The crystal structure of mouse LC3B in complex with the FYCO1 LIR reveals the importance of the flanking region of the LIR motif

2017 ◽  
Vol 73 (3) ◽  
pp. 130-137 ◽  
Author(s):  
Shunya Sakurai ◽  
Taisuke Tomita ◽  
Toshiyuki Shimizu ◽  
Umeharu Ohto
Keyword(s):  
Crystal Structure ◽  
Coiled Coil ◽  
Adaptor Protein ◽  
Structural Basis ◽  
Core Sequence ◽  
Coiled Coil Domain ◽  
Flanking Sequences ◽  
Flanking Region ◽  
Membrane Components ◽  
Lir Motif

FYVE and coiled-coil domain-containing protein 1 (FYCO1), a multidomain autophagy adaptor protein, mediates microtubule plus-end-directed autophagosome transport by interacting with kinesin motor proteins and with the autophagosomal membrane components microtubule-associated protein 1 light chain 3 (LC3), Rab7 and phosphatidylinositol 3-phosphate (PI3P). To establish the structural basis for the recognition of FYCO1 by LC3, the crystal structure of mouse LC3B in complex with the FYCO1 LC3-interacting region (LIR) motif peptide was determined. Structural analysis showed that the flanking sequences N-terminal and C-terminal to the LIR core sequence of FYCO1, as well as the tetrapeptide core sequence, were specifically recognized by LC3B and contributed to the binding. Moreover, comparisons of related structures revealed a conserved mechanism of FYCO1 recognition by different LC3 isoforms among different species.

scholarly journals Structural basis for the interaction of HSP90 with R2TP and TTT complexes

2014 ◽  
Vol 70 (a1) ◽  
pp. C1666-C1666
Author(s):  
Mohinder Pal ◽  
Marc Morgan ◽  
Sarah Phelps ◽  
Mark Roe ◽  
Sarah Parry-Morris ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Crystal Structures ◽  
Structural Basis ◽  
Phosphorylation Sites ◽  
Direct Connection ◽  
Domain Specific ◽  
C Terminus ◽  
Hsp90 Chaperone ◽  
Client Proteins ◽  
Tpr Domain

Assembly and regulation of snoRNPs, RNA polymerases, PI3-kinase-like kinases and the chromatin remodelling complexes, depends on both the TTT complex (Tel2-Tti1-Tti2) and the R2TP complex (Rvb1-Rvb2-Tah1-Pih1p in yeast and RuvBL1-RuvBL2-RPAP3-Pih1D1 in metazoa), which provide the direct connection to Hsp90. Previous studies have shown that the R2TP complex recruits client proteins to Hsp90 for their folding and assembly. In this study, we have determined the crystal structures of three complexes: Hsp90-Tah1-Pih1p, Hsp90-Tah1, Hsp90-RPAP3 (TPR1 and TPR2 domains of RPAP3, each in complex with Hsp90). Tah1 was shown to have an unusual TPR domain, composed of only five α-helices instead of the more usual six or seven. As expected, Tah1 TPR domain binds to the conserved MEEVD motif at the C-terminus of HSP90. In contrast, the C-terminal region of Tah1 is unstructured in the apo form but wraps around the CS domain of Pih1p, thus becoming ordered in the complex, and bridging the interaction between Hsp90 and Pih1p. We show a different modus operandii of Tah1-Hsp90 binding in yeast relative to RPAP3-Hsp90 interactions in metazoa. Finally, we present the crystal structure of the Pih domain of Pih1D1 bound to a phosphopeptide of Tel2 that reveals a novel phosphopeptide-binding domain specific for a subset of CK2 phosphorylation sites. Together these structures define the basis by which the R2TP complex connects the Hsp90 chaperone system to the TTT complex.

Single-component and two-component para -nitrophenol monooxygenases: structural basis for their catalytic difference

2021 ◽  
Author(s):  
Yuan Guo ◽  
De-Feng Li ◽  
Jianting Zheng ◽  
Ying Xu ◽  
Ning-Yi Zhou
Keyword(s):  
Crystal Structure ◽  
Rational Design ◽  
Single Component ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Side Chain ◽  
Amino Acid Residues ◽  
Gram Positive ◽  
Two Component ◽  
Group D

para -Nitrophenol (PNP) is a hydrolytic product of organophosphate insecticides, such as parathion and methylparathion, in soil. Aerobic microbial degradation of PNP has been classically shown to proceed via ‘Hydroquinone (HQ) pathway’ in Gram-negative degraders, whereas via ‘Benzenetriol (BT) pathway’ in Gram-positive ones. ‘HQ pathway’ is initiated by a single-component PNP 4-monooxygenase and ‘BT pathway’ by a two-component PNP 2-monooxygenase. Their rigio-selectivity intrigues us to investigate their catalytic difference through structural study. PnpA1 is the oxygenase component of the two-component PNP 2-monooxygenase from Gram-positive Rhodococcus imtechensis RKJ300. It also catalyzes the hydroxylation of 4-nitrocatechol (4NC) and 2-chloro-4-nitrophenol (2C4NP). However, the mechanisms are unknown. Here, PnpA1 was structurally determined to be a member of group D flavin-dependent monooxygenases with an acyl-CoA dehydrogenase fold. The crystal structure and site-directed mutagenesis underlined the direct involvement of Arg100 and His293 in catalysis. The bulky side chain of Val292 was proposed to push the substrate towards FAD, hence positioning the substrate properly. A variant N450A was found with improved activity for 4NC and 2C4NP, probably because of the reduced steric hindrance. PnpA1 shows obvious difference in substrate selectivity with its close homologues TcpA and TftD, which may be determined by Thr296 and loop 449–454. Above all, our study allows the structural comparison between the two types of PNP monooxygenases. An explanation that accounts for their regio-selectivity was proposed: the different PNP binding manner determines their choice of ortho - or para -hydroxylation on PNP. IMPORTANCE Single-component PNP monoxygenases hydroxylate PNP at 4-position while two-component ones at 2-position. However, their catalytic and structural differences remain elusive. The structure of single-component PNP 4-monooxygenase has previously been determined. In this study, to illustrate their catalytic difference, we resolved the crystal structure of, PnpA1, a typical two-component PNP 2-monooxygenase. The roles of several key amino acid residues in substrate binding and catalysis were revealed and a variant with improved activities towards 4NC and 2C4NP was obtained. Moreover, through comparing the two types of PNP monooxygenases, a hypothesis was proposed to account for their catalytic difference, which gives us a better understanding of these two similar reactions at molecular level. And these results will also be of further aid in enzyme rational design in bioremediation and biosynthesis.

scholarly journals Crystal structure of phosphoribulokinase from Synechococcus sp. strain PCC 6301

2019 ◽  
Vol 75 (4) ◽  
pp. 278-289 ◽  
Author(s):  
Robert H. Wilson ◽  
Manajit Hayer-Hartl ◽  
Andreas Bracher
Keyword(s):  
Crystal Structure ◽  
Disulfide Bond ◽  
Carbon Fixation ◽  
Adaptor Protein ◽  
Structural Basis ◽  
Dimer Interface ◽  
Synechococcus Sp ◽  
Β Sheet ◽  
Loop Residue

Phosphoribulokinase (PRK) catalyses the ATP-dependent phosphorylation of ribulose 5-phosphate to give ribulose 1,5-bisphosphate. Regulation of this reaction in response to light controls carbon fixation during photosynthesis. Here, the crystal structure of PRK from the cyanobacterium Synechococcus sp. strain PCC 6301 is presented. The enzyme is dimeric and has an α/β-fold with an 18-stranded β-sheet at its core. Interestingly, a disulfide bond is found between Cys40 and the P-loop residue Cys18, revealing the structural basis for the redox inactivation of PRK activity. A second disulfide bond appears to rigidify the dimer interface and may thereby contribute to regulation by the adaptor protein CP12 and glyceraldehyde-3-phosphate dehydrogenase.

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