Identification of the structural motif responsible for trimeric assembly of the C-terminal regulatory domains of polycystin channels PKD2L1 and PKD2

2010 ◽  
Vol 429 (1) ◽  
pp. 171-183 ◽  
Author(s):  
Katrina L. Molland ◽  
Anoop Narayanan ◽  
John W. Burgner ◽  
Dinesh A. Yernool
Keyword(s):  
Coiled Coil ◽  
Cd Spectroscopy ◽  
Full Length ◽  
Structural Motif ◽  
Aggregation State ◽  
Regulatory Domains ◽  
Kd Values ◽  
Autosomal Dominant Polycystic Kidney ◽  
Α Helix ◽  
Asymmetric Environment

Polycystin 2-type cation channels PKD2 and PKD2L1 interact with polycystin 1-type proteins PKD1 and PKD1L3 respectively, to form receptor–cation-channel complexes. The PKD2L1–PKD1L3 complex perceives sour taste, whereas disruption of the PKD2–PKD1 complex, responsible for mechanosensation, leads to development of ADPKD (autosomal-dominant polycystic kidney disease). Besides modulating channel activity and related signalling events, the CRDs (C-terminal regulatory domains) of PKD2 and PKD2L1 play a central role in channel oligomerization. The present study investigates the aggregation state of purified full-length PKD2L1-CRD as well as truncations of CRDs from PKD2 channels. Far- and near-UV CD spectroscopy show that the full-length PKD2L1 CRD (PKD2L1-198) and the truncated PKD2 CRD (PKD2-244) are α-helical with no β-sheet, the α-helix content agrees with sequence-based predictions, and some of its aromatic residues are in an asymmetric environment created at least by partially structured regions. Additionally, the CRD truncations exhibit an expected biochemical function by binding Ca2+ in a physiologically relevant range with Kd values of 2.8 μM for PKD2-244 and 0.51 μM for PKD2L1-198. Complimentary biophysical and biochemical techniques establish that truncations of the PKD2 and PKD2L1 CRDs are elongated molecules that assemble as trimers, and the trimeric aggregation state is independent of Ca2+ binding. Finally, we show that a common coiled-coil motif is sufficient and necessary to drive oligomerization of the PKD2 and PKD2L1 CRD truncations under study. Despite the moderate sequence identity (39%) between CRDs of PKD2 and PKD2L1, they both form trimers, implying that trimeric organization of CRDs may be true of all polycystin channels.

scholarly journals Coiled coils as possible models of protein structure evolution

2011 ◽  
Vol 2 (3) ◽  
pp. 199-210 ◽  
Author(s):  
Zoltán Gáspári ◽  
László Nyitray
Keyword(s):  
Large Scale ◽  
Structural Changes ◽  
Coiled Coil ◽  
Coiled Coils ◽  
Structural Motif ◽  
Structure Evolution ◽  
Intrinsically Disordered ◽  
Novel Variants ◽  
Fusion Signal ◽  
Α Helix

AbstractCoiled coils are formed by two or more α-helices wrapped around one another. This structural motif often guides di-, tri- or multimerization of proteins involved in diverse biological processes such as membrane fusion, signal transduction and the organization of the cytoskeleton. Although coiled coil motifs seem conceptually simple and their existence was proposed in the early 1950s, the high variability of the motif makes coiled coil prediction from sequence a difficult task. They might be confused with intrinsically disordered sequences and even more with a recently described structural motif, the charged single α-helix. By contrast, the versatility of coiled coil structures renders them an ideal candidate for protein (re)design and many novel variants have been successfully created to date. In this paper, we review coiled coils in the light of protein evolution by putting our present understanding of the motif and its variants in the context of structural interconversions. We argue that coiled coils are ideal subjects for studies of subtle and large-scale structural changes because of their well-characterized and versatile nature.

The Inhibition of Polysialyltranseferase ST8SiaIV Through Heparin Binding to Polysialyltransferase Domain (PSTD)

2019 ◽  
Vol 15 (5) ◽  
pp. 486-495 ◽  
Author(s):  
Li-Xin Peng ◽  
Xue-Hui Liu ◽  
Bo Lu ◽  
Si-Ming Liao ◽  
Feng Zhou ◽  
...  
Keyword(s):  
Fluorescence Quenching ◽  
Polysialic Acid ◽  
Neuronal Cell ◽  
Cd Spectroscopy ◽  
Migration And Invasion ◽  
Heparin Binding ◽  
Clinical Prognosis ◽  
Quenching Analysis ◽  
Different Types ◽  
Α Helix

Background:The polysialic acid (polySia) is a unique carbohydrate polymer produced on the surface Of Neuronal Cell Adhesion Molecule (NCAM) in a number of cancer cells, and strongly correlates with the migration and invasion of tumor cells and with aggressive, metastatic disease and poor clinical prognosis in the clinic. Its synthesis is catalyzed by two polysialyltransferases (polySTs), ST8SiaIV (PST) and ST8SiaII (STX). Selective inhibition of polySTs, therefore, presents a therapeutic opportunity to inhibit tumor invasion and metastasis due to NCAM polysialylation. Heparin has been found to be effective in inhibiting the ST8Sia IV activity, but no clear molecular rationale. It has been found that polysialyltransferase domain (PSTD) in polyST plays a significant role in influencing polyST activity, and thus it is critical for NCAM polysialylation based on the previous studies.Objective:To determine whether the three different types of heparin (unfractionated hepain (UFH), low molecular heparin (LMWH) and heparin tetrasaccharide (DP4)) is bound to the PSTD; and if so, what are the critical residues of the PSTD for these binding complexes?Methods:Fluorescence quenching analysis, the Circular Dichroism (CD) spectroscopy, and NMR spectroscopy were used to determine and analyze interactions of PSTD-UFH, PSTD-LMWH, and PSTD-DP4.Results:The fluorescence quenching analysis indicates that the PSTD-UFH binding is the strongest and the PSTD-DP4 binding is the weakest among these three types of the binding; the CD spectra showed that mainly the PSTD-heparin interactions caused a reduction in signal intensity but not marked decrease in α-helix content; the NMR data of the PSTD-DP4 and the PSTDLMWH interactions showed that the different types of heparin shared 12 common binding sites at N247, V251, R252, T253, S257, R265, Y267, W268, L269, V273, I275, and K276, which were mainly distributed in the long α-helix of the PSTD and the short 3-residue loop of the C-terminal PSTD. In addition, three residues K246, K250 and A254 were bound to the LMWH, but not to DP4. This suggests that the PSTD-LMWH binding is stronger than the PSTD-DP4 binding, and the LMWH is a more effective inhibitor than DP4.Conclusion:The findings in the present study demonstrate that PSTD domain is a potential target of heparin and may provide new insights into the molecular rationale of heparin-inhibiting NCAM polysialylation.

scholarly journals Coiled-Coil N21 of Hpa1 in Xanthomonas oryzae pv. oryzae Promotes Plant Growth, Disease Resistance and Drought Tolerance in Non-Hosts via Eliciting HR and Regulation of Multiple Defense Response Genes

2020 ◽  
Vol 22 (1) ◽  
pp. 203
Author(s):  
Zhao-Lin Ji ◽  
Mei-Hui Yu ◽  
Ya-Yan Ding ◽  
Jian Li ◽  
Feng Zhu ◽  
...  
Keyword(s):  
Drought Tolerance ◽  
Coiled Coil ◽  
Pharmaceutical Products ◽  
Full Length ◽  
Xanthomonas Oryzae ◽  
Disease Symptoms ◽  
Pathogenic Factors ◽  
Defense Response Genes ◽  
Underlying Mechanisms ◽  
Further Development

Acting as a typical harpin protein, Hpa1 of Xanthomonas oryzae pv. oryzae is one of the pathogenic factors in hosts and can elicit hypersensitive responses (HR) in non-hosts. To further explain the underlying mechanisms of its induced resistance, we studied the function of the most stable and shortest three heptads in the N-terminal coiled-coil domain of Hpa1, named N21Hpa1. Proteins isolated from N21-transgenic tobacco elicited HR in Xanthi tobacco, which was consistent with the results using N21 and full-length Hpa1 proteins expressed in Escherichia coli. N21-expressing tobacco plants showed enhanced resistance to tobacco mosaic virus (TMV) and Pectobacterium carotovora subsp. carotovora (Pcc). Spraying of a synthesized N21 peptide solution delayed the disease symptoms caused by Botrytis cinerea and Monilinia fructicola and promoted the growth and drought tolerance of plants. Further analysis indicated that N21 upregulated the expression of multiple plant defense-related genes, such as genes mediated by salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signaling, and genes related to reactive oxygen species (ROS) biosynthesis. Further, the bioavailability of N21 peptide was better than that of full-length Hpa1Xoo. Our studies support the broad application prospects of N21 peptide as a promising succedaneum to biopesticide Messenger or Illite or other biological pharmaceutical products, and provide a basis for further development of biopesticides using proteins with similar structures.

MODEL POLYPEPTIDES FOR α-HELIX COILED COIL PROTEINS:

2009 ◽  
Vol 10 (5) ◽  
pp. 349-362 ◽  
Author(s):  
L. R. Treiber ◽  
W. Mai Wong ◽  
M. E. Shen ◽  
Dr.A. G. Walton
Keyword(s):  
Coiled Coil ◽  
Α Helix

scholarly journals Microtubule Actin Cross-Linking Factor (Macf)

1999 ◽  
Vol 147 (6) ◽  
pp. 1275-1286 ◽  
Author(s):  
Conrad L. Leung ◽  
Dongming Sun ◽  
Min Zheng ◽  
David R. Knowles ◽  
Ronald K.H. Liem
Keyword(s):  
Calcium Binding ◽  
Coiled Coil ◽  
Specific Protein ◽  
Binding Domain ◽  
Full Length ◽  
Actin Binding ◽  
Cross Linking ◽  
Actin Binding Domain

We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends–PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH2 terminus. However, unlike dystonin, mACF7 does not contain a coiled–coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest–specific protein, Gas2. In this paper, we demonstrate that the NH2-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

scholarly journals Full-length Gαq–phospholipase C-β3 structure reveals interfaces of the C-terminal coiled-coil domain

2013 ◽  
Vol 20 (3) ◽  
pp. 355-362 ◽  
Author(s):  
Angeline M Lyon ◽  
Somnath Dutta ◽  
Cassandra A Boguth ◽  
Georgios Skiniotis ◽  
John J G Tesmer
Keyword(s):  
Phospholipase C ◽  
Coiled Coil ◽  
Full Length ◽  
Coiled Coil Domain

scholarly journals BeStSel: From Secondary Structure Analysis to Protein Fold Prediction by Circular Dichroism Spectroscopy

2020 ◽  
pp. 175-189
Author(s):  
András Micsonai ◽  
Éva Bulyáki ◽  
József Kardos
Keyword(s):  
Circular Dichroism ◽  
Secondary Structure ◽  
Classical Method ◽  
Cd Spectroscopy ◽  
Protein Fold ◽  
Cd Spectra ◽  
Secondary Structure Analysis ◽  
Β Sheets ◽  
Α Helix ◽  
Circular Dichroïsm

Abstract Far-UV circular dichroism (CD) spectroscopy is a classical method for the study of the secondary structure of polypeptides in solution. It has been the general view that the α-helix content can be estimated accurately from the CD spectra. However, the technique was less reliable to estimate the β-sheet contents as a consequence of the structural variety of the β-sheets, which is reflected in a large spectral diversity of the CD spectra of proteins containing this secondary structure component. By taking into account the parallel or antiparallel orientation and the twist of the β-sheets, the Beta Structure Selection (BeStSel) method provides an improved β-structure determination and its performance is more accurate for any of the secondary structure types compared to previous CD spectrum analysis algorithms. Moreover, BeStSel provides extra information on the orientation and twist of the β-sheets which is sufficient for the prediction of the protein fold. The advantage of CD spectroscopy is that it is a fast and inexpensive technique with easy data processing which can be used in a wide protein concentration range and under various buffer conditions. It is especially useful when the atomic resolution structure is not available, such as the case of protein aggregates, membrane proteins or natively disordered chains, for studying conformational transitions, testing the effect of the environmental conditions on the protein structure, for verifying the correct fold of recombinant proteins in every scientific fields working on proteins from basic protein science to biotechnology and pharmaceutical industry. Here, we provide a brief step-by-step guide to record the CD spectra of proteins and their analysis with the BeStSel method.

scholarly journals Ligand-induced conformational rearrangements regulate the switch between membrane-proximal and distal functions of Rho kinase 2

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
István Hajdú ◽  
András Szilágyi ◽  
Barbara M. Végh ◽  
András Wacha ◽  
Dániel Györffy ◽  
...  
Keyword(s):  
Structural Model ◽  
Coiled Coil ◽  
Rho Kinase ◽  
Mechanistic Explanation ◽  
Kinase Domain ◽  
Multifunctional Protein ◽  
Regulatory Domains ◽  
Extended Conformation ◽  
Activity Measurements ◽  
Present Solution

AbstractRho-associated protein kinase 2 (ROCK2) is a membrane-anchored, long, flexible, multidomain, multifunctional protein. Its functions can be divided into two categories: membrane-proximal and membrane-distal. A recent study concluded that membrane-distal functions require the fully extended conformation, and this conclusion was supported by electron microscopy. The present solution small-angle X-ray scattering (SAXS) study revealed that ROCK2 population is a dynamic mixture of folded and partially extended conformers. Binding of RhoA to the coiled-coil domain shifts the equilibrium towards the partially extended state. Enzyme activity measurements suggest that the binding of natural protein substrates to the kinase domain breaks up the interaction between the N-terminal kinase and C-terminal regulatory domains, but smaller substrate analogues do not. The present study reveals the dynamic behaviour of this long, dimeric molecule in solution, and our structural model provides a mechanistic explanation for a set of membrane-proximal functions while allowing for the existence of an extended conformation in the case of membrane-distal functions.

Cation channel regulation by COOH-terminal cytoplasmic tail of polycystin-1: mutational and functional analysis

2002 ◽  
Vol 8 (2) ◽  
pp. 87-98 ◽  
Author(s):  
David H. Vandorpe ◽  
Sabine Wilhelm ◽  
Lianwei Jiang ◽  
Oxana Ibraghimov-Beskrovnaya ◽  
Marina N. Chernova ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Point Mutations ◽  
Coiled Coil ◽  
Cytoplasmic Tail ◽  
Surface Expression ◽  
Dominant Negative ◽  
Cation Channel ◽  
Loss Of Function ◽  
Cation Current ◽  
Autosomal Dominant Polycystic Kidney

Polycystin-1 (PKD1) mutations account for ∼85% of autosomal dominant polycystic kidney disease (ADPKD). We have shown previously that oocyte surface expression of a transmembrane fusion protein encoding part of the cytoplasmic COOH terminus of PKD1 increases activity of a Ca2+-permeable cation channel. We show here that human ADPKD mutations incorporated into this fusion protein attenuated or abolished encoded cation currents. Point mutations and truncations showed that cation current expression requires integrity of a region encompassing the putative coiled coil domain of the PKD1 cytoplasmic tail. Whereas these loss-of-function mutants did not exhibit dominant negative phenotypes, coexpression of a fusion protein expressing the interacting COOH-terminal cytoplasmic tail of PKD2 did suppress cation current. Liganding of the ectodomain of the PKD1 fusion protein moderately activated cation current. The divalent cation permeability and pharmacological profile of the current has been extended. Inducible expression of the PKD1 fusion in EcR-293 cells was also associated with activation of cation channels and increased Ca2+ entry.

scholarly journals Human ADPKD primary cyst epithelial cells with a novel, single codon deletion in the PKD1 gene exhibit defective ciliary polycystin localization and loss of flow-induced Ca2+ signaling

2007 ◽  
Vol 292 (3) ◽  
pp. F930-F945 ◽  
Author(s):  
Chang Xu ◽  
Sandro Rossetti ◽  
Lianwei Jiang ◽  
Peter C. Harris ◽  
Ursa Brown-Glaberman ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Nk Cells ◽  
Coiled Coil ◽  
Human Kidney ◽  
Coiled Coil Domain ◽  
Normal Human Kidney ◽  
Cyst Cells ◽  
Localization Signal ◽  
Normal Human ◽  
Autosomal Dominant Polycystic Kidney

Autosomal dominant polycystic kidney disease (ADPKD) gene products polycystin-1 (PC1) and polycystin-2 (PC2) colocalize in the apical monocilia of renal epithelial cells. Mouse and human renal cells without PC1 protein show impaired ciliary mechanosensation, and this impairment has been proposed to promote cystogenesis. However, most cyst epithelia of human ADPKD kidneys appear to express full-length PC1 and PC2 in normal or increased abundance. We show that confluent primary ADPKD cyst cells with the novel PC1 mutation ΔL2433 and with normal abundance of PC1 and PC2 polypeptides lack ciliary PC1 and often lack ciliary PC2, whereas PC1 and PC2 are both present in cilia of confluent normal human kidney (NK) epithelial cells in primary culture. Confluent NK cells respond to shear stress with transient increases in cytoplasmic Ca2+ concentration ([Ca2+]i), dependent on both extracellular Ca2+ and release from intracellular stores. In contrast, ADPKD cyst cells lack flow-sensitive [Ca2+]i signaling and exhibit reduced endoplasmic reticulum Ca2+ stores and store-depletion-operated Ca2+ entry but retain near-normal [Ca2+]i responses to ANG II and to vasopressin. Expression of wild-type and mutant CD16.7-PKD1(115–226) fusion proteins reveals within the COOH-terminal 112 amino acids of PC1 a coiled-coil domain-independent ciliary localization signal. However, the coiled-coil domain is required for CD16.7-PKD1(115–226) expression to accelerate decay of the flow-induced Ca2+ signal in NK cells. These data provide evidence for ciliary dysfunction and polycystin mislocalization in human ADPKD cells with normal levels of PC1.

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