Evolutionary approach to identify the potential surface of Isd11 involved in interactions with Nfs1.

<p>Sequence logos of the Isd11 (top) and other members of the LYR families. The letter size is proportional to the degree of conservation of each residue in the multiple alignment of the family. The residues that are most specifically conserved in the Isd11 subfamily and less so in other LYR proteins are indicated with arrows. The ISD11 logo is based on an alignment of 211 Isd11 orthologues with maximally 90% identity and is limited to positions that are present in yeast Isd11. Identification of residues specific to Isd11 is based on a comparison with other members of the LYR family: LYRM2, LYRM3, LYRM5, LYRM7 and ACN9.</p

Structural characterization of ISD11.

<p>A) Far-UV CD spectrum of ISD11. B) Thermal denaturation curve of ISD11 after cleavage of the GST tag monitored at the CD signal at 220 nm. The data were fitted to a two state transition. C) Projection of a ¹⁵N-NOESY-HSQC spectrum of ISD11 recorded at 800 MHz with a mixing time of 450 ms. All nitrogen planes are displayed.</p

ISD11 forms a complex with IscS.

<p>A) Overlay of SOFAST-HMQC spectra of 34 μM [U-¹⁵N] labelled ISD11 titrated with increasing molar equivalence (eq) of unlabelled IscS. The experiment was recorded at constant ISD11 concentration. Each spectrum had to be recorded over 2h to achieve a reasonable signal-to-noise ratio. B) Analytical SEC using a 10/300 Superdex 200 increase column for the NMR sample in A) of ISD11:IscS at 1:4 molar ratio and of ISD11 and IscS separately. C), D) and E) SDS-PAGE analysis of the fractions from the SEC in (B) for ISD11 (C), IscS (D) and ISD11 titrated with 4 molar equivalents of IscS (D). Bands corresponding to ISD11 and IscS are indicated with the symbols * and • respectively.</p

IscS co-purifies with ISD11.

<p>A) Chromatograms measured at A₂₈₀ and A₃₉₂ for the purification of ISD11 by SEC on a 16/600 Superdex 75 column. B) SDS-PAGE of SEC peaks I, II and III from A). C) Absorbance spectrum of co-purified peak I from A).</p

Characterization of the ISD11-IscS interaction.

<p>A) Analytical size exclusion chromatograms at 280 nm of 30 μM of either His₁₀-tagged IscS, of coexpressed ISD11-His₆ (pZM6) and IscS (pSL219), and separately purified IscS-His₁₀ (30 μM) and ISD11 (150 μM, purified by GST-tag) mixed in a ratio of 1 to 5. B) L-cysteine desulfurase activity as determined by the methylene blue assay. The activity of separately purified IscS-His and IscS-His mixed with ISD11, raitio 1:4, (pZM96) was compared. The assay contained 2 mM DTT, 100 μM L-cysteine, 2 μM IscS-His and 8 μM ISD11. The assay was carried out for 5 min at 37°C. The absorbance of formed methylene blue was determined at 670 nm.</p

Oligomeric state of ISD11.

<p>A) Chromatograms from analytical SEC on a 10/300 Superdex 75 column for Ubiquitin, ISD11, holo-Ferredoxin (Fdx), Ferredoxin-NADP Reductase (FNR) and bovine serum albumin (BSA). B) SEC/MALLS analysis of ISD11 at concentrations of 1 mg/mL and 2 mg/mL on a 10/300 Superdex 200 increase column. Chromatograms are output from dRI detector. Points are weight-averaged molar mass calculated at 1 s intervals throughout elution of chromatographic peaks.</p

A Unique Spumavirus Gag N-terminal Domain with Functional Properties of Orthoretroviral Matrix and Capsid

<div><p>The <i>Spumaretrovirinae</i>, or foamyviruses (FVs) are complex retroviruses that infect many species of monkey and ape. Although FV infection is apparently benign, trans-species zoonosis is commonplace and has resulted in the isolation of the Prototypic Foamy Virus (PFV) from human sources and the potential for germ-line transmission. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. In addition, PFV Gag interacts with the FV Envelope (Env) protein to facilitate budding of infectious particles. Presently, there is a paucity of structural information with regards FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. Therefore, in order to probe the functional overlap of FV and orthoretroviral Gag and learn more about FV egress and replication we have undertaken a structural, biophysical and virological study of PFV-Gag. We present the crystal structure of a dimeric amino terminal domain from PFV, Gag-NtD, both free and in complex with the leader peptide of PFV Env. The structure comprises a head domain together with a coiled coil that forms the dimer interface and despite the shared function it is entirely unrelated to either the capsid or matrix of Gag from other retroviruses. Furthermore, we present structural, biochemical and virological data that reveal the molecular details of the essential Gag-Env interaction and in addition we also examine the specificity of Trim5α restriction of PFV. These data provide the first information with regards to FV structural proteins and suggest a model for convergent evolution of <i>gag</i> genes where structurally unrelated molecules have become functionally equivalent.</p></div

Particle production and infectivity of PFV-Gag central domain mutants.

<p>(<b>A</b>) Western blot analysis of producer cell lysates (<b>Cell</b>) and pelleted viral supernatants (<b>Virus</b>) with polyclonal antibodies specific for PFV-Gag (α-Gag) and PFV Env-LP (α-Env-LP) or monoclonal antibodies specific for PFV-PR/RT (α-PR/RT) and integrase (α-IN). Residue substitutions in Gag are indicated above each track, (<i>wt</i>) wild type virus, (<i>wt +iRT</i>) wild type virus with defective reverse transcriptase. In the right-hand panel %<i>wt</i> are different <i>wt</i> control loadings and arrows indicate the migration of Gag, Env and Pol proteins. (<b>B</b>) Relative amounts of released Gag quantified from Western blots data from two independent experiments. (<b>C</b>) Relative infectivity of extracellular 293T cell culture supernatants using an eGFP marker gene transfer assay, determined 3 days post infection. Means and standard deviations of three independent experiments are shown. The values obtained using the wild type Gag packaging vector were arbitrarily set to 100%. Absolute titres of these supernatants were 1.8 x 10⁶ to 1.1 x 10⁷ ffu/ml.</p

NMR structure of PFV-Gag CtD_CEN homodimer.

<p>(<b>A</b>) Cartoon representation of the structure of the PFV-Gag CtD_CEN dimer. Monomer-A is shown in dark blue and Monomer-B in cyan. The α-helices are labelled as for PFV-Gag(300–477) and the N- and C-termini of each monomer are indicated. (<b>B</b>) Details of the homodimer interface. Residues that contribute to the interface are shown as sticks. (<b>C</b>) C(S) distribution derived from sedimentation velocity data recorded from PFV-Gag CtD_CEN L410E/M413E mutant at 2 mg/mL (dashed line). The C(S) distribution derived from sedimentation velocity data recorded from <i>wt</i> PFV-Gag CtD_CEN at 2 mg /mL is shown also for comparison (solid line).</p

NMR and refinement statistics for PFV-Gag Central domains.

<p>NMR and refinement statistics for PFV-Gag Central domains.</p