Structural Origin of the High Affinity of a Chemically Evolved Lanthanide‐Binding Peptide

Nitz, Mark; Sherawat, Manashi; Franz, Katherine J.; Peisach, Ezra; Allen, Karen N.; Imperiali, Barbara

doi:10.1002/anie.200460028

Cited by 169 publications

(252 citation statements)

References 24 publications

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“…1A). Because the LBT-Tb 3+ emission decay has a well-defined time constant of ∼2.4 ms, the donor-only (DO) emission (i.e., the donor emission in the absence of the acceptor) is very specific and distinguishable from background (13,18,19). Also, because the LBT is incorporated into the backbone of the protein, the donor becomes tied to the structure of the channel, likely decreasing the uncertainty of the donor position.…”

Section: +mentioning

confidence: 99%

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β1-subunit–induced structural rearrangements of the Ca ²⁺ - and voltage-activated K ⁺ (BK) channel

Castillo

Sánchez-Rodríguez

Hyde

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Large-conductance Ca 2+ -and voltage-activated K + (BK) channels are involved in a large variety of physiological processes. Regulatory β-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α-or β1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) β1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the β1-subunit within the α/β1-subunit complex.lanthanide resonance energy transfer | BK channels | β1-subunit I mportant physiological processes involve Ca 2+ entry into cells mediated by voltage-dependent Ca 2+ channels. This divalent cation influx is essential for life because it permits, for example, the adequate functioning of smooth muscle or neurosecretion to occur. Some mechanism must be put into action, however, to control Ca 2+ influx, either to dampen or to stop the physiological effects of the cytoplasmic increase in Ca 2+. In many cases, this dampening mechanism is accomplished by one of the most broadly expressed channels in mammals: the large-conductance Ca 2+ -and voltage-activated K + (BK) channel (1-3). Because there is a single gene coding for the BK channel (Slowpoke KNCMA1), channel diversity must be a consequence of alternative splicing and/or interaction with regulatory subunits. In fact, both mechanisms account for BK channel diversity, but the most dramatic changes in BK channel properties are brought about through the interaction with regulatory subunits, membrane-integral proteins, denominated BK β-subunits (β1-β4) (4-7) and the recently discovered γ-subunits (γ1-γ4) (8, 9).Structurally, the BK channel is a homotetramer of its poreforming α-subunit and is a member of the voltage-dependent potassium (Kv) channel family. Distinct from Kv channels, however, BK channel subunits are composed of seven transmembrane domains S0-S6 (10, 11). Little is known about the detailed structure of the membrane-spanning portion of the BK channel, or of the α/β1-subunit complex. Here, we used a variant of Förster resonance energy transfer (FRET), called lanthanide-based resonance energy transfer (LRET), to determine the positions of the N terminus (NT) and S0, S1, and S2 transmembrane segments of the α-subunit of the BK channel, as well as the position of the β1-subunit in the α/β1-subunit complex. LRET uses luminescent lanthanides (e.g., Tb 3+ ) as donor instead of conventional fluorophores. This technique has been successfully used to measure intramolecular distances and to...

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Section: +mentioning

confidence: 99%

“…Because Tb 3+ has a spiked emission spectrum, it is possible to isolate the sensitized emission (SE) of the acceptor with relative ease by using an adequate optical filter. Additionally, we used the genetically encoded lanthanide-binding tag (LBT) to chelate Tb 3+ donor within the protein structure (18) (Fig. 1A).…”

mentioning

confidence: 99%

β1-subunit–induced structural rearrangements of the Ca ²⁺ - and voltage-activated K ⁺ (BK) channel

Castillo

Sánchez-Rodríguez

Hyde

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…LRET is a FRET technique that takes advantage of the properties of lanthanides, particularly Tb 3+ , which has a long lifetime; dark regions between emission peaks in its spectrum; and, most importantly, isotropic emission (20). Briefly, the donor is Tb 3+ -bound to a genetically encoded LBT motif with the sequence YIDTNNDGWYEGDELLA, which binds Tb 3+ with high affinity (K d = 57 nM) (42). For these experiments, we used a modified LBT that has an additional tryptophan (underlined), YWDTNNDGWYEGDELLA, to improve the Tb 3+ D signal of Nav-LBT constructs (Fig.…”

Section: Methodsmentioning

confidence: 99%

Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET

Kubota

Durek

Dang

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: β-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.V oltage-gated sodium channels (Navs) play an essential role in the generation and propagation of action potentials in excitable cells (1). Eukaryotic Navs are composed of a poreforming α subunit and auxiliary β subunits. The α subunit is a large single-polypeptide chain organized in four different domains (DI-DIV), each of which has a voltage-sensing domain (VSD; S1-S4 segments) and a pore-forming domain (S5-S6 segments). Each domain has a different amino acid composition, pointing to some level of functional specialization. Site-directed fluorimetry shows that the VSDs in DI, DII, and DIII of the rat skeletal muscle voltage-gated sodium channel (Nav1.4) are activated by depolarization faster than in DIV (2). From this observation, it has been hypothesized that DI-III VSDs control the pore opening of the mammalian Nav, whereas the DIV VSD governs its fast inactivation (2-5).Although mammalian Nav function has been studied comprehensively, the precise structural basis for the gating mechanisms has not been fully elucidated. The crystal structures of several prokaryotic Navs have been solved recently; however, in contrast to the mammalian Navs, they are homotetrameric, and thus structurally more closely related to the organization of voltage-gated potassium channels (Kvs) (6-9). The structure of a human L-type voltage-gated calcium channel type 1.1 (Cav1.1), which is a large single polypeptide composed of four different domains similar to mammalian Navs, has been resolved using cryoelectron microscopy (10, 11). However, functional studies have shown that gating mechanisms of mammalian Cav channels are indeed different from gating mechanisms in mammalian Navs (12). Furthermore, such structural studies only provide static snapshots of the channels in one of many possible conformational states. Therefore, techniques that provide dynamic structural information are nee...

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“…The introduction of partial order by slight molecular alignment will result nonzero RDC observables. This partial order can be introduced by exploiting the inherent magnetic anisotropic susceptibility of the molecule [1], incorporating artificial tags with high magnetic anisotropic susceptibility [24], or using a liquid crystalline media [25]. Once restored, RDCs can be measured relatively easily and represent an abundant source of highly precise information on parameters such as the relative orientations of different inter-nuclear 'bonds' within the molecule or internal motion.…”

Section: Residual Dipolar Couplingmentioning

confidence: 99%

REDCRAFT: A tool for simultaneous characterization of protein backbone structure and motion from RDC data

Bryson

Tian

Prestegard

et al. 2008

Journal of Magnetic Resonance

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REDCRAFT, a new open source software tool that accommodates the analysis of RDC data for simultaneous structure and dynamics characterization of proteins is presented in this article. Simultaneous consideration of structure and motion is believed to be necessary for accurate representation of the solution-state of a protein. REDCRAFT is designed to primarily utilize RDC data from multiple alignment media in two stages. During Stage-I, a list of possible torsion angles joining any two neighboring peptide planes is ranked based on their fitness to experimental constraints; in Stage-II, a dipeptide fragment is extended by addition of one peptide plane at a time. The algorithm adopted by REDCRAFT is very efficient and can produce a structure for an 80 residue protein within two hours on a typical desktop computer. REDCRAFT exhibits robustness with respect to noise and missing data. REDCRAFT describes the overall alignment of the molecule in the form of an order tensor matrix and is capable of identifying peptide fragments with internal dynamics. Identification of the location of internal motion will permit a more accurate structural representation. Experimental data from two proteins as well as simulated data are presented to illustrate the capabilities of REDCRAFT in both structure determination and identification of the dynamical regions. Keywordssimultaneous; backbone; protein; structure; motion; residual; dipolar; dynamics IntroductionStructural elucidation, including complete structures of individual domains of proteins as well as the assembly of biomolecular complexes, is often a requisite step in understanding fundamental physiological processes, or in the design of drugs to combat a disease. Therefore, the development of methods leading to rapid, cost-effective structure elucidation is an important task. In addition, it is important to develop methods that can simultaneously deal with internal motion in these assemblies. Motions on a physiologically relevant time scale have been suggested to play an important role in the biological function [6;7]. Traditionally, Correspondence to: Homayoun Valafar. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript characterization of inter-molecular dynamics has been separated from structure elucidation, increasing the cost and time of these studies. Furthermore, conceptually, it is difficult to separate structure from dynamics since observables used for structure determination are perturbed by motion, and therefore any attempt at struct...

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Structural Origin of the High Affinity of a Chemically Evolved Lanthanide‐Binding Peptide

Cited by 169 publications

References 24 publications

β1-subunit–induced structural rearrangements of the Ca ²⁺ - and voltage-activated K ⁺ (BK) channel

β1-subunit–induced structural rearrangements of the Ca ²⁺ - and voltage-activated K ⁺ (BK) channel

Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET

REDCRAFT: A tool for simultaneous characterization of protein backbone structure and motion from RDC data

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Structural Origin of the High Affinity of a Chemically Evolved Lanthanide‐Binding Peptide

Cited by 169 publications

References 24 publications

β1-subunit–induced structural rearrangements of the Ca 2+ - and voltage-activated K + (BK) channel

β1-subunit–induced structural rearrangements of the Ca 2+ - and voltage-activated K + (BK) channel

Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET

REDCRAFT: A tool for simultaneous characterization of protein backbone structure and motion from RDC data

Contact Info

Product

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β1-subunit–induced structural rearrangements of the Ca ²⁺ - and voltage-activated K ⁺ (BK) channel

β1-subunit–induced structural rearrangements of the Ca ²⁺ - and voltage-activated K ⁺ (BK) channel