scholarly journals Crystal Structure of a ligand-bound LacY–Nanobody Complex

2018 ◽  
Vol 115 (35) ◽  
pp. 8769-8774 ◽  
Author(s):  
Hemant Kumar ◽  
Janet S. Finer-Moore ◽  
Xiaoxu Jiang ◽  
Irina Smirnova ◽  
Vladimir Kasho ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Lactose Permease ◽  
Transmembrane Helices ◽  
Helical Bundle ◽  
Flexible Loop ◽  
Sugar Binding ◽  
Open Conformation ◽  
Substrate Analog ◽  
Multiple Conformations ◽  
Alternating Access

The lactose permease of Escherichia coli (LacY), a dynamic polytopic membrane transport protein, catalyzes galactoside/H+ symport and operates by an alternating access mechanism that exhibits multiple conformations, the distribution of which is altered by sugar-binding. Camelid nanobodies were made against a double-mutant Gly46 → Trp/Gly262 → Trp (LacYWW) that produces an outward-open conformation, as opposed to the cytoplasmic open-state crystal structure of WT LacY. Nanobody 9047 (Nb9047) stabilizes WT LacY in a periplasmic-open conformation. Here, we describe the X-ray crystal structure of a complex between LacYWW, the high-affinity substrate analog 4-nitrophenyl-α-d-galactoside (NPG), and Nb9047 at 3-Å resolution. The present crystal structure demonstrates that Nb9047 binds to the periplasmic face of LacY, primarily to the C-terminal six-helical bundle, while a flexible loop of the Nb forms a bridge between the N- and C-terminal halves of LacY across the periplasmic vestibule. The bound Nb partially covers the vestibule, yet does not affect the on-rates or off-rates for the substrate binding to LacYWW, which implicates dynamic flexibility of the Nb–LacYWW complex. Nb9047-binding neither changes the overall structure of LacYWW with bound NPG, nor the positions of side chains comprising the galactoside-binding site. The current NPG-bound structure exhibits a more occluded periplasmic vestibule than seen in a previous structure of a (different Nb) apo-LacYWW/Nb9039 complex that we argue is caused by sugar-binding, with major differences located at the periplasmic ends of transmembrane helices in the N-terminal half of LacY.

scholarly journals Crystal structure of a LacY–nanobody complex in a periplasmic-open conformation

2016 ◽  
Vol 113 (44) ◽  
pp. 12420-12425 ◽  
Author(s):  
Xin Jiang ◽  
Irina Smirnova ◽  
Vladimir Kasho ◽  
Jianping Wu ◽  
Kunio Hirata ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Free Access ◽  
Lactose Permease ◽  
Sugar Binding ◽  
Open Conformation ◽  
Trp Mutant ◽  
Multiple Conformations ◽  
Polytopic Membrane Protein ◽  
Alternating Access

The lactose permease of Escherichia coli (LacY), a dynamic polytopic membrane protein, catalyzes galactoside–H+ symport and operates by an alternating access mechanism that exhibits multiple conformations, the distribution of which is altered by sugar binding. We have developed single-domain camelid nanobodies (Nbs) against a mutant in an outward (periplasmic)-open conformation to stabilize this state of the protein. Here we describe an X-ray crystal structure of a complex between a double-Trp mutant (Gly46→Trp/Gly262→Trp) and an Nb in which free access to the sugar-binding site from the periplasmic cavity is observed. The structure confirms biochemical data indicating that the Nb binds stoichiometrically with nanomolar affinity to the periplasmic face of LacY primarily to the C-terminal six-helix bundle. The structure is novel because the pathway to the sugar-binding site is constricted and the central cavity containing the galactoside-binding site is empty. Although Phe27 narrows the periplasmic cavity, sugar is freely accessible to the binding site. Remarkably, the side chains directly involved in binding galactosides remain in the same position in the absence or presence of bound sugar.

scholarly journals Transient conformers of LacY are trapped by nanobodies

2015 ◽  
Vol 112 (45) ◽  
pp. 13839-13844 ◽  
Author(s):  
Irina Smirnova ◽  
Vladimir Kasho ◽  
Xiaoxu Jiang ◽  
Els Pardon ◽  
Jan Steyaert ◽  
...  
Keyword(s):  
Lactose Permease ◽  
Conformational States ◽  
Dynamic Membrane ◽  
Conformational Selection ◽  
Sugar Binding ◽  
Open Conformation ◽  
Transport Cycle ◽  
Fluorescence Change ◽  
Alternating Access ◽  
Selection Of

The lactose permease of Escherichia coli (LacY), a highly dynamic membrane protein, catalyzes symport of a galactopyranoside and an H+ by using an alternating access mechanism, and the transport cycle involves multiple conformational states. Single-domain camelid nanobodies (Nbs) developed against a LacY mutant immobilized in an outward (periplasmic)-open conformation bind to the flexible WT protein and stabilize the open-outward conformation(s). Here, we use site-directed, distance-dependent Trp quenching/unquenching of fluorescent probes inserted on opposite surfaces of LacY to assess the conformational states of the protein complexed with each of eight unique Nbs that bind exclusively to the periplasmic side and block transport, but increase the accessibility of the sugar-binding site. Nb binding involves conformational selection of LacY molecules with exposed binding epitopes. Each of eight Nbs induces quenching with three pairs of cytoplasmic Trp/fluorophore probes, indicating closing of cytoplasmic cavity. In reciprocal fashion, the same Nbs induce unquenching of fluorescence in three pairs of periplasmic probes due to opening of the periplasmic cavity. Because the extent of fluorescence change with various Nbs differs and the differences correlate with changes in the rate of sugar binding, it is also concluded that the Nbs stabilize several different outward-open conformations of LacY.

scholarly journals Engineered occluded apo-intermediate of LacY

2018 ◽  
Vol 115 (50) ◽  
pp. 12716-12721 ◽  
Author(s):  
Irina Smirnova ◽  
Vladimir Kasho ◽  
H. Ronald Kaback
Keyword(s):  
Binding Site ◽  
Lactose Permease ◽  
Binding Studies ◽  
X Ray Crystallography ◽  
Sugar Binding ◽  
Transport Cycle ◽  
Open Structures ◽  
Unrestricted Access ◽  
Tight Association ◽  
Alternating Access

The lactose permease of Escherichia coli (LacY) utilizes an alternating access symport mechanism with multiple conformational intermediates, but only inward (cytoplasmic)- or outward (periplasmic)-open structures have been characterized by X-ray crystallography. It is demonstrated here with sugar-binding studies that cross-linking paired-Cys replacements across the closed cytoplasmic cavity stabilize an occluded conformer with an inaccessible sugar-binding site. In addition, a nanobody (Nb) that stabilizes a periplasmic-open conformer with an easily accessible sugar-binding site in WT LacY fails to cause the cytoplasmic cross-linked mutants to become accessible to galactoside, showing that the periplasmic cavity is closed. These results are consistent with tight association of the periplasmic ends in two pairs of helices containing clusters of small residues in the packing interface between N- and C-terminal six-helix bundles of the symporter. However, after reduction of the disulfide bond, the Nb markedly increases the rate of galactoside binding, indicating unrestricted access to the Nb epitope and the galactoside-binding site from the periplasm. The findings indicate that the cross-linked cytoplasmic double-Cys mutants resemble an occluded apo-intermediate in the transport cycle.

scholarly journals A chemiosmotic mechanism of symport

2015 ◽  
Vol 112 (5) ◽  
pp. 1259-1264 ◽  
Author(s):  
H. Ronald Kaback
Keyword(s):  
Driving Forces ◽  
Lactose Permease ◽  
Transmembrane Helices ◽  
Electrochemical Gradient ◽  
Induced Fit ◽  
X Ray ◽  
Membrane Transport Proteins ◽  
Limiting Step ◽  
Experimental Findings ◽  
Alternating Access

Lactose permease (LacY), a paradigm for the largest family of membrane transport proteins, catalyzes the coupled translocation of a galactoside and an H+ across the Escherichia coli membrane (galactoside/H+ symport). Initial X-ray structures reveal N- and C-terminal domains, each with six largely irregular transmembrane helices surrounding an aqueous cavity open to the cytoplasm. Recently, a structure with a narrow periplasmic opening and an occluded galactoside was obtained, confirming many observations and indicating that sugar binding involves induced fit. LacY catalyzes symport by an alternating access mechanism. Experimental findings garnered over 45 y indicate the following: (i) The limiting step for lactose/H+ symport in the absence of the H+ electrochemical gradient (∆µ̃H+) is deprotonation, whereas in the presence of ∆µ̃H+, the limiting step is opening of apo LacY on the other side of the membrane; (ii) LacY must be protonated to bind galactoside (the pK for binding is ∼10.5); (iii) galactoside binding and dissociation, not ∆µ̃H+, are the driving forces for alternating access; (iv) galactoside binding involves induced fit, causing transition to an occluded intermediate that undergoes alternating access; (v) galactoside dissociates, releasing the energy of binding; and (vi) Arg302 comes into proximity with protonated Glu325, causing deprotonation. Accumulation of galactoside against a concentration gradient does not involve a change in Kd for sugar on either side of the membrane, but the pKa (the affinity for H+) decreases markedly. Thus, transport is driven chemiosmotically but, contrary to expectation, ∆µ̃H+ acts kinetically to control the rate of the process.

scholarly journals Structures of plasmepsin II fromPlasmodium falciparumin complex with two hydroxyethylamine-based inhibitors

2015 ◽  
Vol 71 (12) ◽  
pp. 1531-1539 ◽  
Author(s):  
Rosario Recacha ◽  
Janis Leitans ◽  
Inara Akopjana ◽  
Lilija Aprupe ◽  
Peteris Trapencieris ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Complex Structure ◽  
Antimalarial Agents ◽  
Flexible Loop ◽  
Open Conformation ◽  
Active Site Cavity ◽  
Plasmepsin Ii ◽  
Complex Structural ◽  
New Compounds

Plasmepsin II (PMII) is one of the ten plasmepsins (PMs) identified in the genome ofPlasmodium falciparum, the causative agent of the most severe and deadliest form of malaria. Owing to the emergence ofP. falciparumstrains that are resistant to current antimalarial agents such as chloroquine and sulfadoxine/pyrimethamine, there is a constant pressure to find new and lasting chemotherapeutic drug therapies. Previously, the crystal structure of PMII in complex with NU655, a potent antimalarial hydroxyethylamine-based inhibitor, and the design of new compounds based on it have been reported. In the current study, two of these newly designed hydroxyethylamine-based inhibitors, PG418 and PG394, were cocrystallized with PMII and their structures were solved, analyzed and compared with that of the PMII–NU655 complex. Structural analysis of the PMII–PG418 complex revealed that the flap loop can adopt a fully closed conformation, stabilized by interactions with the inhibitor, and a fully open conformation, causing an overall expansion in the active-site cavity, which in turn causes unstable binding of the inhibitor. PG418 also stabilizes the flexible loop Gln275–Met286 of another monomer in the asymmetric unit of PMII, which is disordered in the PMII–NU655 complex structure. The crystal structure of PMII in complex with the inhibitor PG418 demonstrates the conformational flexibility of the active-site cavity of the plasmepsins. The interactions of the different moieties in the P1′ position of PG418 and PG394 with Thr217 have to be taken into account in the design of new potent plasmepsin inhibitors.

The lactose permease of Escherichia coli : overall structure, the sugar-binding site and the alternating access model for transport

FEBS Letters ◽  
2003 ◽  
Vol 555 (1) ◽  
pp. 96-101 ◽  
Author(s):  
Jeff Abramson ◽  
Irina Smirnova ◽  
Vladimir Kasho ◽  
Gillian Verner ◽  
So Iwata ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Lactose Permease ◽  
Sugar Binding ◽  
Alternating Access ◽  
Access Model

scholarly journals GapR binds DNA through dynamic opening of its tetrameric interface

2020 ◽  
Vol 48 (16) ◽  
pp. 9372-9386
Author(s):  
Qian Huang ◽  
Bo Duan ◽  
Xianzhi Dong ◽  
Shilong Fan ◽  
Bin Xia
Keyword(s):  
Crystal Structure ◽  
Caulobacter Crescentus ◽  
Structural Rearrangement ◽  
Exchange Equilibrium ◽  
Base Pairs ◽  
Open Conformation ◽  
Multiple Conformations ◽  
Dynamic Exchange ◽  
Dna Complex ◽  
Major Conformation

Abstract GapR is a nucleoid-associated protein that is an essential regulator of chromosome replication in the cell cycle model Caulobacter crescentus. Here, we demonstrate that free GapR is a homotetramer, but not a dimer as previously reported (Guo et al., Cell 175: 583–597, 2018). We have determined the crystal structure of GapR in complex with a 10-bp A-tract DNA, which has an open tetrameric conformation, different from the closed clamp conformation in the previously reported crystal structure of GapR/DNA complex. The free GapR adopts multiple conformations in dynamic exchange equilibrium, with the major conformation resembling the closed tetrameric conformation, while the open tetrameric conformation is a representative of minor conformers. As it is impossible for the circular genomic DNA to get into the central DNA binding tunnel of the major conformation, we propose that GapR initially binds DNA through the open conformation, and then undergoes structural rearrangement to form the closed conformation which fully encircles the DNA. GapR prefers to bind DNA with 10-bp consecutive A/T base pairs nonselectively (Kd ∼12 nM), while it can also bind GC-rich DNA sequence with a reasonable affinity of about 120 nM. Besides, our results suggest that GapR binding results in widening the minor groove of AT-rich DNA, instead of overtwisting DNA.

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