Identification of an alternating-access dynamics mutant of EmrE with impaired transport

Proteins that perform active transport must alternate the access of a binding site, first to one side of a membrane and then to the other, resulting in the transport of bound substrates across the membrane. To better understand this process, we sought to identify mutants of the small multidrug resistance transporter EmrE with reduced rates of alternating access. We performed extensive scanning mutagenesis by changing every amino acid residue to Val, Ala, or Gly, and then screening the drug resistance phenotypes of the resulting mutants. We identified EmrE mutants that had impaired transport activity but retained the ability to bind substrate and further tested their alternating access rates using NMR. Ultimately, we were able to identify a single mutation, S64V, which significantly reduced the rate of alternating access but did not impair substrate binding. Six other transport-impaired mutants did not have reduced alternating access rates, highlighting the importance of other aspects of the transport cycle to achieve drug resistance activity in vivo. To better understand the transport cycle of EmrE, efforts are now underway to determine a high-resolution structure using the S64V mutant identified here

Structure-Guided Evolution of Potent and Selective CHK1 Inhibitors through Scaffold Morphing

Pyrazolopyridine inhibitors with low micromolar potency for CHK1 and good selectivity against CHK2 were previously identified by fragment-based screening. The optimization of the pyrazolopyridines to a series of potent and CHK1-selective isoquinolines demonstrates how fragment-growing and scaffold morphing strategies arising from a structure-based understanding of CHK1 inhibitor binding can be combined to successfully progress fragment-derived hit matter to compounds with activity in vivo. The challenges of improving CHK1 potency and selectivity, addressing synthetic tractability, and achieving novelty in the crowded kinase inhibitor chemical space were tackled by multiple scaffold morphing steps, which progressed through tricyclic pyrimido[2,3-b]azaindoles to N-(pyrazin-2-yl)pyrimidin-4-amines and ultimately to imidazo[4,5-c]pyridines and isoquinolines. A potent and highly selective isoquinoline CHK1 inhibitor (SAR-020106) was identified, which potentiated the efficacies of irinotecan and gemcitabine in SW620 human colon carcinoma xenografts in nude mice

Structures of an Apo and a Binary Complex of an Evolved Archeal B Family DNA Polymerase Capable of Synthesising Highly Cy-Dye Labelled DNA

Thermophilic DNA polymerases of the polB family are of great importance in biotechnological applications including high-fidelity PCR. Of particular interest is the relative promiscuity of engineered versions of the exo- form of polymerases from the Thermo- and Pyrococcales families towards non-canonical substrates, which enables key advances in Next-generation sequencing. Despite this there is a paucity of structural information to guide further engineering of this group of polymerases. Here we report two structures, of the apo form and of a binary complex of a previously described variant (E10) of Pyrococcus furiosus (Pfu) polymerase with an ability to fully replace dCTP with Cyanine dye-labeled dCTP (Cy3-dCTP or Cy5-dCTP) in PCR and synthesise highly fluorescent "CyDNA" densely decorated with cyanine dye heterocycles. The apo form of Pfu-E10 closely matches reported apo form structures of wild-type Pfu. In contrast, the binary complex (in the replicative state with a duplex DNA oligonucleotide) reveals a closing movement of the thumb domain, increasing the contact surface with the nascent DNA duplex strand. Modelling based on the binary complex suggests how bulky fluorophores may be accommodated during processive synthesis and has aided the identification of residues important for the synthesis of unnatural nucleic acid polymers.status: publishe

A structural model for maturation of the hepatitis B virus core

Hepatitis B virus, a widespread and serious human pathogen, replicates by reverse transcription of an RNA intermediate. The virus consists of an inner nucleocapsid or core, surrounded by a lipid envelope containing virally encoded surface proteins. Using electron cryomicroscopy, we compare the structures of the bacterially expressed RNA-containing core particle and the mature DNA-containing core particle extracted from virions. We show that the mature core contains 240 subunits in a T = 4 arrangement similar to that in expressed core (T is the triangulation number and the icosahedral shell contains 60 T subunits). During the infective cycle, the core assembles in an immature state around a complex of viral pregenomic RNA and polymerase. After reverse transcription with concomitant degradation of the RNA, the now mature core buds through a cellular membrane containing the surface proteins to become enveloped. Envelopment must not happen before reverse transcription is completed, so it has been hypothesized that a change in capsid structure may signal maturation. Our results show significant differences in structure between the RNA- and DNA-containing cores. One such difference is in a hydrophobic pocket, formed largely from residues that, on mutation, lead to abnormal secretion. We suggest that the changes we see are related to maturation and control of envelopment, and we propose a mechanism based on DNA synthesis for their triggering. © 2005 by The National Academy of Sciences of the USA

Structures of an Apo and a Binary Complex of an Evolved Archeal B Family DNA Polymerase Capable of Synthesising Highly Cy-Dye Labelled DNA

<div><p>Thermophilic DNA polymerases of the polB family are of great importance in biotechnological applications including high-fidelity PCR. Of particular interest is the relative promiscuity of engineered versions of the exo- form of polymerases from the <i>Thermo-</i> and <i>Pyrococcales</i> families towards non-canonical substrates, which enables key advances in Next-generation sequencing. Despite this there is a paucity of structural information to guide further engineering of this group of polymerases. Here we report two structures, of the apo form and of a binary complex of a previously described variant (E10) of <i>Pyrococcus furiosus</i> (Pfu) polymerase with an ability to fully replace dCTP with Cyanine dye-labeled dCTP (Cy3-dCTP or Cy5-dCTP) in PCR and synthesise highly fluorescent “CyDNA” densely decorated with cyanine dye heterocycles. The apo form of Pfu-E10 closely matches reported apo form structures of wild-type Pfu. In contrast, the binary complex (in the replicative state with a duplex DNA oligonucleotide) reveals a closing movement of the thumb domain, increasing the contact surface with the nascent DNA duplex strand. Modelling based on the binary complex suggests how bulky fluorophores may be accommodated during processive synthesis and has aided the identification of residues important for the synthesis of unnatural nucleic acid polymers.</p></div

Structural similarity of Apo Pfu-E10 with other polymerases.

1<p>The RMSd was calculated using Superpose from the entire length of the Pfu-E10 except where stated.</p>2<p>ClustalW2 was used for obtaining % identity score.</p

Data collection and refinement statistics.

1<p>Values in parentheses are for the highest resolution bin (2.53–2.40 Å and 2.46–2.40 Å for the apo Pfu-E10 for scaling and refinement respectively, with 2.98–2.9 Å and 3.06–2.90 Å for the Pfu-E10:DNA complex).</p

Structure of the Pfu-E10:DNA binary complex.

<p>Cartoon representation of the binary complex<b>.</b> Polymerase domains are coloured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070892#pone-0070892-g003" target="_blank">Figure 3</a>. The DNA primer strand is shown in grey, the template strand in magenta.</p

Polymerase activity at 60°C.

<p>A) The primer extension assays used a primer labelled with FAM (fluorescein) and unlabelled template. Sites of Cy5-dCTP incorporation (Gs in the template) are shown in red. B) Primer extension time course comparing wild-type Pfu(exo-) and engineered Pfu-E10 polymerases at 60°C. Extension times are shown in minutes. Extension products used to quantify extension beyond the seven consecutive Cy5-dCTP incorporations (C₇ challenge) are highlighted in red – see Materials and Methods for details. C) Fraction of the primers extended beyond the C₇ challenge for both tested polymerases – results are shown for two independent experiments.</p