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Modern approaches in metallodrug research focus on compounds that bind protein targets rather than DNA. However, the identification of protein targets and binding sites is challenging. Using intact mass spectrometry and proteomics, we investigated the binding of the antimetastatic agent RAPTA-C to the model proteins ubiquitin, cytochrome c, lysozyme, and myoglobin. Binding to cytochrome c and lysozyme was negligible. However, ubiquitin bound up to three Ru moieties, two of which were localized at Met1 and His68 as [Ru(cym)], and [Ru(cym)] or [Ru(cym)(PTA)] adducts, respectively. Myoglobin bound up to four [Ru(cym)(PTA)] moieties and five sites were identified at His24, His36, His64, His81/82 and His113. Collision-induced unfolding (CIU) studies via ion-mobility mass spectrometry allowed measuring protein folding as a function of collisional activation. CIU of protein-RAPTA-C adducts showed binding of [Ru(cym)] to Met1 caused a significant compaction of ubiquitin, likely from N-terminal S-Ru-N chelation, while binding of [Ru(cym)(PTA)] to His residues of ubiquitin or myoglobin induced a smaller effect. Interestingly, the folded state of ubiquitin formed by His functionalization was more stable than Met1 metalation. The data suggests that selective metalation of amino acids at different positions on the protein impacts the conformation and potentially the biological activity of anticancer compounds., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Mass spectrometry (MS) is an analytical technique for molecule identification that can be used for investigating protein-metal complex interactions. Once the MS data is collected, the mass spectra are usually interpreted manually to identify the adducts formed as a result of the interactions between proteins and metal-based species. However, with increasing resolution, dataset size, and species complexity, the time required to identify adducts and the error-prone nature of manual assignment have become limiting factors in MS analysis. AdductHunter is a open-source web-based analysis tool that  automates the peak identification process using constraint integer optimization to find feasible combinations of protein and fragments, and dynamic time warping to calculate the dissimilarity between the theoretical isotope pattern of a species and its experimental isotope peak distribution. Empirical evaluation on a collection of 22 unique MS datasetsshows fast and accurate identification of protein-metal complex adducts in deconvoluted mass spectra., (© 2024. The Author(s).)

Optimization of compound 11L led to the identification of novel HIV capsid modulators, quinazolin-4-one-bearing phenylalanine derivatives, displaying potent antiviral activities against both HIV-1 and HIV-2. Notably, derivatives 12a2 and 21a2 showed significant improvements, with 2.5-fold over 11L and 7.3-fold over PF74 for HIV-1, and approximately 40-fold over PF74 for HIV-2. The X-ray co-crystal structures confirmed the multiple pocket occupation of 12a2 and 21a2 in the binding site. Mechanistic studies revealed a dual-stage inhibition profile, where the compounds disrupted capsid-host factor interactions at the early stage and promoted capsid misassembly at the late stage. Remarkably, 12a2 and 21a2 significantly promoted capsid misassembly, outperforming 11L, PF74, and LEN. The substitution of easily metabolized amide bond with quinolin-4-one marginally enhanced the stability of 12a2 in human liver microsomes compared to controls. Overall, 12a2 and 21a2 highlight their potential as potent HIV capsid modulators, paving the way for future advancements in anti-HIV drug design.

3,6,7-trimethyllumazine (Lepteridine™) is a newly discovered natural pteridine derivative unique to Mānuka ( Leptospermum scoparium ) nectar and honey, with no previously reported biological activity. Pteridine derivative-based medicines, such as methotrexate, are used to treat auto-immune and inflammatory diseases, and Mānuka honey reportedly possesses anti-inflammatory properties and is used topically as a wound dressing. MMP-9 is a potential candidate protein target as it is upregulated in recalcitrant wounds and intestinal inflammation. Using gelatin zymography, 40 μg/mL Lepteridine TM inhibited the gelatinase activities of both pro- (22%, p < 0.0001) and activated (59%, p < 0.01) MMP-9 forms. By comparison, Lepteridine TM exerted modest (~10%) inhibition against a chromogenic peptide substrate and no effect against a fluorogenic peptide substrate. These findings suggest that Lepteridine TM may not interact within the catalytic domain of MMP-9 and exerts a negligible effect on the active site hydrolysis of small soluble peptide substrates. Instead, the findings implicate fibronectin II domain interactions by Lepteridine TM which impair gelatinase activity, possibly through perturbed tethering of MMP-9 to the gelatin matrix. Molecular modelling analyses were equivocal over interactions at the S1' pocket versus the fibronectin II domain, while molecular dynamic calculations indicated rapid exchange kinetics. No significant degradation of synthetic or natural Lepteridine TM in Mānuka honey occurred during simulated gastrointestinal digestion. MMP-9 regulates skin and gastrointestinal inflammatory responses and extracellular matrix remodelling. These results potentially implicate Lepteridine TM bioactivity in Mānuka honey's reported beneficial effects on wound healing via topical application and anti-inflammatory actions in gastrointestinal disorder models via oral consumption.

Trichomonas vaginalis is a human protozoan parasite that causes trichomoniasis, a prevalent sexually transmitted infection. Trichomoniasis is accompanied by a shift to a dysbiotic vaginal microbiome that is depleted of lactobacilli. Studies on co-cultures have shown that vaginal bacteria in eubiosis (e.g. Lactobacillus gasseri) have antagonistic effects on T. vaginalis pathogenesis, suggesting that the parasite might benefit from shaping the microbiome to dysbiosis (e.g. Gardnerella vaginalis among other anaerobes). We have recently shown that T. vaginalis has acquired NlpC/P60 genes from bacteria, expanding them to a repertoire of nine TvNlpC genes in two distinct clans, and that TvNlpCs of clan A are active against bacterial peptidoglycan. Here, we expand this characterization to TvNlpCs of clan B. In this study, we show that the clan organisation of NlpC/P60 genes is a feature of other species of Trichomonas, and that Histomonas meleagridis has sequences related to one clan. We characterized the 3D structure of TvNlpC&#95;B3 alone and with the inhibitor E64 bound, probing the active site of these enzymes for the first time. Lastly, we demonstrated that TvNlpC&#95;B3 and TvNlpC&#95;B5 have complementary activities with the previously described TvNlpCs of clan A and that exogenous expression of these enzymes empower this mucosal parasite to take over populations of vaginal lactobacilli in mixed cultures. TvNlpC&#95;B3 helps control populations of L. gasseri, but not of G. vaginalis, which action is partially inhibited by E64. This study is one of the first to show how enzymes produced by a mucosal protozoan parasite may contribute to a shift on the status of a microbiome, helping explain the link between trichomoniasis and vaginal dysbiosis. Further understanding of this process might have significant implications for treatments in the future., Competing Interests: The authors have no competing interest., (Copyright: © 2023 Barnett et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Pt(terpyridine) complexes are well-known DNA intercalators. The introduction of an NHC co-ligand rendered such a complex highly antiproliferative in cancer cells compared to its chlorido derivative. Despite the high potency, zebrafish embryos tolerated the compound well, especially compared to cisplatin. DNA interaction studies support a mode of action related to intercalation.

Flowering of the reference legume Medicago truncatula is promoted by winter cold (vernalization) followed by long-day photoperiods (VLD) similar to winter annual Arabidopsis. However, Medicago lacks FLC and CO, key regulators of Arabidopsis VLD flowering. Most plants have two INHIBITOR OF GROWTH (ING) genes (ING1 and ING2), encoding proteins with an ING domain with two anti-parallel alpha-helices and a plant homeodomain (PHD) finger, but their genetic role has not been previously described. In Medicago, Mting1 gene-edited mutants developed and flowered normally, but an Mting2-1 Tnt1 insertion mutant and gene-edited Mting2 mutants had developmental abnormalities including delayed flowering particularly in VLD, compact architecture, abnormal leaves with extra leaflets but no trichomes, and smaller seeds and barrels. Mting2 mutants had reduced expression of activators of flowering, including the FT-like gene MtFTa1, and increased expression of the candidate repressor MtTFL1c, consistent with the delayed flowering of the mutant. MtING2 overexpression complemented Mting2-1, but did not accelerate flowering in wild type. The MtING2 PHD finger bound H3K4me2/3 peptides weakly in vitro, but analysis of gene-edited mutants indicated that it was dispensable to MtING2 function in wild-type plants. RNA sequencing experiments indicated that >7000 genes are mis-expressed in the Mting2-1 mutant, consistent with its strong mutant phenotypes. Interestingly, ChIP-seq analysis identified >5000 novel H3K4me3 locations in the genome of Mting2-1 mutants compared to wild type R108. Overall, our mutant study has uncovered an important physiological role of a plant ING2 gene in development, flowering, and gene expression, which likely involves an epigenetic mechanism., (© 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Transcription of endogenous retroviral elements are tightly regulated during development by members of the KRAB-containing zinc finger proteins (KRAB-ZFPs) and the co-repressor Trim28 (also known as Kap-1 or Tif1β). KRAB-ZFPs form the largest family of transcription regulators in mammals and initiate transcriptional silencing by tethering Trim28 to a target locus. Subsequently, Trim28 recruits chromatin modifying effectors resulting in the formation of heterochromatin. In the present study, we identify surface exposed residues on the central six turns of the Trim28 coiled-coil region forming the binding interface for the KRAB domain. Using AlphaFold2 (AF2) we provide high confidence models of the interface between Trim28 and the KRAB domain and identified leucine 301 on each chain of the Trim28 monomer to act as a pin extending into a hydrophobic pocket on the KRAB domain surface. Site directed mutations in the Trim28-KRAB binding interface abolished binding to the KRAB domain. Our work provides a detailed understanding of the specific interactions between the KRAB domain and the Trim28 coiled-coil and how this interaction may be regulated during silencing events., (© 2022 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

N-Heterocyclic carbene (NHC) ligands are widely investigated in medicinal inorganic chemistry. Here, we report the preparation and characterization of a series of half-sandwich [M(L)(NHC)Cl 2 ] (M = Ru, Os, Rh, Ir; L = cym/Cp*) complexes with a N-flanking anthracenyl moiety attached to imidazole- and benzimidazole-derived NHC ligands. The anticancer activity of the complexes was investigated in cell culture studies where, in comparison to a Rh derivative with an all-carbon-donor-atom-based ligand ( 5a ), they were found to be cytotoxic with IC 50 values in the low micromolar range. The Ru derivative 1a was chosen as a representative for stability studies as well as for biomolecule interaction experiments. It underwent partial chlorido/aqua ligand exchange in DMSO- d 6 /D 2 O to rapidly form an equilibrium in aqueous media. The reactions of 1a with biomolecules proceeded quickly and resulted in the formation of adducts with amino acids, DNA, and protein. Hen egg white lysozyme crystals were soaked with 1a , and the crystallographic analysis revealed an interaction with an l-aspartic acid residue (Asp119), resulting in the cleavage of the p -cymene ligand but the retention of the NHC moiety. Cell morphology studies for the Rh analog 3a suggested that the cytotoxicity is exerted via mechanisms different from that of cisplatin.

Metal complexes can be considered a "paradigm of promiscuity" when it comes to their interactions with proteins. They often form adducts with a variety of donor atoms in an unselective manner. We have characterized the adducts formed between a series of isostructural N-heterocyclic carbene (NHC) complexes with Ru, Os, Rh, and Ir centers and the model protein hen egg white lysozyme by X-ray crystallography and mass spectrometry. Distinctive behavior for the metal compounds was observed with the more labile Ru and Rh complexes targeting mainly a surface l-histidine moiety through cleavage of p-cymene or NHC co-ligands, respectively. In contrast, the more inert Os and Ir derivatives were detected abundantly in an electronegative binding pocket after undergoing ligand exchange of a chlorido ligand for an amino acid side chain. Computational studies supported the binding profiles and hinted at the role of the protein microenvironment for metal complexes eliciting selectivity for specific binding sites on the protein., (© 2021 Wiley-VCH GmbH.)

The paramyxoviral phosphoprotein (P protein) is the non-catalytic subunit of the viral RNA polymerase, and coordinates many of the molecular interactions required for RNA synthesis. All paramyxoviral P proteins oligomerize via a centrally located coiled-coil that is connected to a downstream binding domain by a dynamic linker. The C-terminal region of the P protein coordinates interactions between the catalytic subunit of the polymerase, and the viral nucleocapsid housing the genomic RNA. The inherent flexibility of the linker is believed to facilitate polymerase translocation. Here we report biophysical and structural characterization of the C-terminal region of the P protein from Menangle virus (MenV), a bat-borne paramyxovirus with zoonotic potential. The MenV P protein is tetrameric but can dissociate into dimers at sub-micromolar protein concentrations. The linker is globally disordered and can be modeled effectively as a worm-like chain. However, NMR analysis suggests very weak local preferences for alpha-helical and extended beta conformation exist within the linker. At the interface between the disordered linker and the structured C-terminal binding domain, a gradual disorder-to-order transition occurs, with X-ray crystallographic analysis revealing a dynamic interfacial structure that wraps the surface of the binding domain.

The development of bifunctional platinum complexes with the ability to interact with DNA via different binding modes is of interest in anticancer metallodrug research. Therefore, we report platinum(II) terpyridine complexes to target DNA by coordination and/or through a tethered alkylating moiety. The platinum complexes were evaluated for their in vitro antiproliferative properties against the human cancer cell lines HCT116 (colorectal), SW480 (colon), NCI-H460 (non-small cell lung), and SiHa (cervix) and generally exhibited potent antiproliferative activity although lower than their respective terpyridine ligands. 1 H NMR spectroscopy and/or ESI-MS studies on the aqueous stability and reactivity with various small biomolecules, acting as protein and DNA model compounds, were used to establish potential modes of action for these complexes. These investigations indicated rapid binding of complex PtL3 to the biomolecules through coordination to the Pt center, while PtL4 in addition alkylated 9-ethylguanine. PtL3 was investigated for its reactivity to the model protein hen egg white lysozyme (HEWL) by protein crystallography which allowed identification of the N δ1 atom of His15 as the binding site.

The life cycle of Baculoviridae family insect viruses depends on the viral protein kinase, PK-1, to phosphorylate the regulatory protein, p6.9, to induce baculoviral genome release. Here, we report the crystal structure of Cydia pomenella granulovirus PK-1, which, owing to its likely ancestral origin among host cell AGC kinases, exhibits a eukaryotic protein kinase fold. PK-1 occurs as a rigid dimer, where an antiparallel arrangement of the αC helices at the dimer core stabilizes PK-1 in a closed, active conformation. Dimerization is facilitated by C-lobe:C-lobe and N-lobe:N-lobe interactions between protomers, including the domain-swapping of an N-terminal helix that crowns a contiguous β-sheet formed by the two N-lobes. PK-1 retains a dimeric conformation in solution, which is crucial for catalytic activity. Our studies raise the prospect that parallel, side-to-side dimeric arrangements that lock kinase domains in a catalytically-active conformation could function more broadly as a regulatory mechanism among eukaryotic protein kinases.

While most Rh-N-heterocyclic carbene (NHC) complexes currently investigated in anticancer research contain a Rh(III) metal center, an increasing amount of research is focusing on the cytotoxic activity and mode of action of square-planar [RhCl(COD)(NHC)] (where COD = 1,5-cyclooctadiene) which contains a Rh(I) center. The enzyme thioredoxin reductase (TrxR) and the protein albumin have been proposed as potential targets, but the molecular processes taking place upon protein interaction remain elusive. Herein, we report the preparation of peptide-conjugated and its nonconjugated parent [RhCl(COD)(NHC)] complexes, an in-depth investigation of both their stability in solution, and a crystallographic study of protein interaction. The organorhodium compounds showed a rapid loss of the COD ligand and slow loss of the NHC ligand in aqueous solution. These ligand exchange reactions were reflected in studies on the interaction with hen egg white lysozyme (HEWL) as a model protein in single-crystal X-ray crystallographic investigations. Upon treatment of HEWL with an amino acid functionalized [RhCl(COD)(NHC)] complex, two distinct rhodium adducts were found initially after 7 d of incubation at His15 and after 4 weeks also at Lys33. In both cases, the COD and chlorido ligands had been substituted with aqua and/or hydroxido ligands. While the histidine (His) adduct also indicated a loss of the NHC ligand, the lysine (Lys) adduct retained the NHC core derived from the amino acid l-histidine. In either case, an octahedral coordination environment of the metal center indicates oxidation to Rh(III). This investigation gives the first insight on the interaction of Rh(I)(NHC) complexes and proteins at the molecular level.

Members of the TRIM protein family have been shown to inhibit a range of viral infections. Recently, TRIM69 was identified as a potent inhibitor of Vesicular stomatitis Indiana virus infection, with its inhibition being dependent upon multimerization. Using SEC-MALLS analysis, it is demonstrated that the assembly of TRIM69 is mediated through the RING domain and not the Bbox domain as has been shown for other TRIM proteins. Using X-ray crystallography, the structure of the TRIM69 RING domain has been determined to a resolution of 2.1 Å, the oligomerization interface has been identified and regions outside the four-helix bundle have been observed to form interactions that are likely to support assembly.

Metal complexes provide a versatile platform to develop novel anticancer pharmacophores, and they form stable compounds with N -heterocyclic carbene (NHC) ligands, some of which have been shown to inhibit the cancer-related selenoenzyme thioredoxin reductase (TrxR). To expand a library of isostructural NHC complexes, we report here the preparation of Rh III - and Ir III (Cp*)(NHC)Cl 2 (Cp* = η 5 -pentamethylcyclopentadienyl) compounds and comparison of their properties to the Ru II - and Os II (cym) analogues (cym = η 6 - p -cymene). Like the Ru II - and Os II (cym) complexes, the Rh III - and Ir III (Cp*) derivatives exhibit cytotoxic activity with half maximal inhibitory concentration (IC 50 ) values in the low micromolar range against a set of four human cancer cell lines. In studies on the uptake and localization of the compounds in cancer cells by X-ray fluorescence microscopy, the Ru and Os derivatives were shown to accumulate in the cytoplasmic region of treated cells. In an attempt to tie the localization of the compounds to the inhibition of the tentative target TrxR, it was surprisingly found that only the Rh complexes showed significant inhibitory activity at IC 50 values of ∼1 μM, independent of the substituents on the NHC ligand. This indicates that, although TrxR may be a potential target for anticancer metal complexes, it is unlikely the main target or the sole target for the Ru, Os, and Ir compounds described here, and other targets should be considered. In contrast, Rh(Cp*)(NHC)Cl 2 complexes may be a scaffold for the development of TrxR inhibitors.

The HML2 (HERV-K) group constitutes the most recently acquired family of human endogenous retroviruses, with many proviruses less than one million years old. Many maintain intact open reading frames and provirus expression together with HML2 particle formation are observed in early stage human embryo development and are associated with pluripotency as well as inflammatory disease, cancers and HIV-1 infection. Here, we reconstruct the core structural protein (CA) of an HML2 retrovirus, assemble particles in vitro and employ single particle cryogenic electron microscopy (cryo-EM) to determine structures of four classes of CA Fullerene shell assemblies. These icosahedral and capsular assemblies reveal at high-resolution the molecular interactions that allow CA to form both pentamers and hexamers and show how invariant pentamers and structurally plastic hexamers associate to form the unique polyhedral structures found in retroviral cores.

Cisplatin and its second and third generation analogues are widely used in the treatment of cancer. To study their reactions with proteins, we present a method based on SDS-PAGE separation and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) for platinum detection in the reaction between human serum albumin (HSA) and cisplatin. We developed matrix-matched standards of HSA/cisplatin mixtures and used them to quantify the amount of adducts formed at different HSA:cisplatin ratios. We noted that cisplatin incubation with HSA resulted in the formation of higher order HSA n-mers, depending on the amount of cisplatin added. This caused a depletion of the HSA dimer bands, while the majority of HSA was present as the monomer. Inducing the formation of such higher molecular weight species may have an impact on the mode of action of metallodrugs., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

TRIM28 (also known as KAP1 or TIF1β) is the universal co-repressor of the Krüppel-associated box-containing zinc finger proteins (Krab-ZFPs), the largest family of transcription factors in mammals. During early embryogenesis, TRIM28 mediates the transcriptional silencing of many endogenous retroviral elements and genomic imprinted sites. Silencing is initiated by the recruitment of TRIM28 to a target locus by members of the Krab-ZFP. Subsequently, TRIM28 functions as a scaffold protein to recruit chromatin modifying effectors featuring SETDB1, HP1 and the NuRD complex. Although many protein partners involved in silencing have been identified, the molecular basis of the protein interactions that mediate silencing remains largely unclear. In the present study, we identified the first Bbox domain (T28&#95;B1 135-203) as a molecular interface responsible for the formation of higher-order oligomers of TRIM28. The structure of this domain reveals a new interface on the surface of the Bbox domain. Mutants disrupting the interface disrupt the formation of oligomers but have no observed effect on transcriptional silencing defining a single TRIM28 dimer as the functional unit for silencing. Using assembly-deficient mutants, we employed small-angle X-ray scattering and biophysical techniques to characterize binding to member of the Krab-ZFP family. This allows us to narrow and define the binding interface to the center of the coiled-coil region (residues 294-321) of TRIM28 and define mutants that abolish binding to the Krab-ZFP proteins., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Many viral genomes encode kinase and phosphatase enzymes to manipulate pathways that are controlled by phosphorylation events. The majority of viral phosphatase genes occur in the Baculoviridae and Poxviridae families of large DNA viruses. The corresponding protein sequences belong to four major homology groups, and structures are currently available for only two of these. Here, the first structure from the third group, the protein tyrosine phosphatase-2 (PTP-2) class of viral phosphatases, is described. It is shown that Cydia pomonella granulovirus PTP-2 has the same general fold and active-site architecture as described previously for other phosphatases, in the absence of significant sequence homology. Additionally, it has a novel C-terminal extension in an area corresponding to the interface of dimeric poxvirus phosphatases belonging to the Tyr-Ser protein phosphatase homology group.

Correction for 'Unexpected arene ligand exchange results in the oxidation of an organoruthenium anticancer agent: the first X-ray structure of a protein-Ru(carbene) adduct' by Matthew P. Sullivan et al., Chem. Commun., 2018, 54, 6120-6123.

The human eukaryotic pathogen Trichomonas vaginalis causes trichomoniasis, a prevalent sexually transmitted infection. This extracellular protozoan is intimately associated with the human vaginal mucosa and microbiota, but key aspects of the complex interactions between the parasite and the vaginal bacteria remain elusive. We report that T. vaginalis has acquired, by lateral gene transfer from bacteria, genes encoding peptidoglycan hydrolases of the NlpC/P60 family. Two of the T. vaginalis enzymes were active against bacterial peptidoglycan, retaining the active-site fold and specificity as dl-endopeptidases. The endogenous NlpC/P60 genes are transcriptionally upregulated in T. vaginalis in the presence of bacteria. The overexpression of an exogenous copy enables the parasite to outcompete bacteria from mixed cultures, consistent with the biochemical activity of the enzyme. Our study results highlight the relevance of the interactions of this eukaryotic pathogen with bacteria, a poorly understood aspect of the biology of this important human parasite. IMPORTANCE Trichomonas vaginalis is a parasitic protozoan of the human urogenital tract that causes trichomoniasis, a very common sexually transmitted disease. Despite residing extracellularly and in close association with the vaginal bacteria (i.e., the microbiota), very little is known about the nature of the parasite-bacterium interactions. Our study showed that this parasite had acquired genes from bacteria which retained their original function. They produce active enzymes capable of degrading peptidoglycan, a unique polymer of the bacterial cell envelope, helping the parasite to outcompete bacteria in mixed cultures. This study was the first to show that a laterally acquired group of genes enables a eukaryotic mucosal pathogen to control bacterial population. We highlight the importance of understanding the interactions between pathogens and microbiota, as the outcomes of these interactions are increasingly understood to have important implications on health and disease., (Copyright © 2018 Pinheiro et al.)

The retroviral restriction factor tripartite motif-containing 5α (Trim5α) acts during the early postentry stages of the retroviral life cycle to block infection by a broad range of retroviruses, disrupting reverse transcription and integration. The mechanism of this restriction is poorly understood, but it has recently been suggested to involve recruitment of components of the autophagy machinery, including members of the mammalian autophagy-related 8 (ATG8) family involved in targeting proteins to the autophagosome. To better understand the molecular details of this interaction, here we utilized analytical ultracentrifugation to characterize the binding of six ATG8 isoforms and determined the crystal structure of the Trim5α Bbox coiled-coil region in complex with one member of the mammalian ATG8 proteins, autophagy-related protein LC3 B (LC3B). We found that Trim5α binds all mammalian ATG8s and that, unlike the typical LC3-interacting region (LIR) that binds to mammalian ATG8s through a β-strand motif comprising approximately six residues, LC3B binds to Trim5α via the α-helical coiled-coil region. The orientation of the structure demonstrated that LC3B could be accommodated within a Trim5α assembly that can bind the retroviral capsid. However, mutation of the binding interface does not affect retroviral restriction. Comparison of the typical linear β-strand LIR with our atypical helical LIR reveals a conservation of the presentation of residues that are required for the interaction with LC3B. This observation expands the range of LC3B-binding proteins to include helical binding motifs and demonstrates a link between Trim5α and components of the autophagosome., (© 2018 Keown et al.)

The first X-ray structures of adducts formed between a RuII(N-heterocyclic carbene)(η6-p-cymene) compound and a protein are reported. Coordination to the protein induced the cleavage of the cymene ligand and EPR spectroscopy demonstrated the oxidation of the Ru centre.

Aerobic life brings with it a need to respond to external redox stress in ways that preserve key processes. S uppressor of c opper s ensitivity (Scs) proteins contribute to this response in some bacteria, but have poorly defined molecular functions. Furlong et al. now demonstrate that two Scs proteins from Proteus mirabilis provide a redox relay functionally equivalent to, but structurally distinct from, the Dsb proteins that orchestrate disulfide bonding in Escherichia coli , emphasizing the wide prevalence of this mechanism in bacteria., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2018 Goldstone and Baker.)

Peroxiredoxins are abundant peroxidase enzymes that are key regulators of the cellular redox environment. A major subgroup of these proteins, the typical 2-Cys peroxiredoxins, can switch between dimers and decameric or dodecameric rings, during the catalytic cycle. The necessity of this change in quaternary structure for function as a peroxidase is not fully understood. In order to explore this, human peroxiredoxin 3 (Prx3) protein was engineered to form both obligate dimers (S75E Prx3) and stabilised dodecameric rings (S78C Prx3), uncoupling structural transformations from the catalytic cycle. The obligate dimer, S75E Prx3, retained catalytic activity towards hydrogen peroxide, albeit significantly lower than the wildtype and S78C proteins, suggesting an evolutionary advantage of having higher order self-assemblies., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Dihydrodipicolinate reductase (DHDPR) catalyses the second reaction in the diaminopimelate pathway of lysine biosynthesis in bacteria and plants. In contrast with the tetrameric bacterial DHDPR enzymes, we show that DHDPR from Vitis vinifera (grape) and Selaginella moellendorffii are dimeric in solution. In the present study, we have also determined the crystal structures of DHDPR enzymes from the plants Arabidopsis thaliana and S. moellendorffii , which are the first dimeric DHDPR structures. The analysis of these models demonstrates that the dimer forms through the intra-strand interface, and that unique secondary features in the plant enzymes block tetramer assembly. In addition, we have also solved the structure of tetrameric DHDPR from the pathogenic bacteria Neisseria meningitidis Measuring the activity of plant DHDPR enzymes showed that they are much more prone to substrate inhibition than the bacterial enzymes, which appears to be a consequence of increased flexibility of the substrate-binding loop and higher affinity for the nucleotide substrate. This higher propensity to substrate inhibition may have consequences for ongoing efforts to increase lysine biosynthesis in plants., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Metalation of hen egg white lysozyme (HEWL) with organometallics was studied with physicochemical methods in solid state, solution and the gas phase. While metalation did not affect the crystal structure of HEWL significantly, protein destabilisation was detected in gas phase and solution.

With the aim of increasing the accumulation of Ru anticancer agents in the tumor, a targeted delivery strategy based on a maleimide anchor for the biological vector human serum albumin (HSA) was developed. A group of piano stool Ru- and Os(η 6 -arene) complexes carrying a maleimide-functionalized N-phenyl-2-pyridinecarbothioamide (PCA) ligand was designed allowing for covalent conjugation to biological thiols. The complexes were characterized by NMR spectroscopy, ESI-MS, elemental analysis and single-crystal X-ray diffraction analysis. The compounds were shown to undergo halido/aqua ligand exchange reactions in aqueous solution, depending mainly on the metal center and the nature of the halide. In vitro cytotoxicity studies revealed low potency which is explained by the observed high reactivity of the maleimide to the thiol of l-cysteine (Cys), while the metal center itself shows little affinity to amino acids of the model protein lysozyme., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The Spumaretrovirinae, or foamy viruses (FVs) are complex retroviruses that infect many species of monkey and ape. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. However, there is a paucity of structural information for FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. To probe the functional overlap of FV and orthoretroviral Gag we have determined the structure of a central region of Gag from the Prototype FV (PFV). The structure comprises two all α-helical domains NtDCEN and CtDCEN that although they have no sequence similarity, we show they share the same core fold as the N- (NtDCA) and C-terminal domains (CtDCA) of archetypal orthoretroviral capsid protein (CA). Moreover, structural comparisons with orthoretroviral CA align PFV NtDCEN and CtDCEN with NtDCA and CtDCA respectively. Further in vitro and functional virological assays reveal that residues making inter-domain NtDCEN-CtDCEN interactions are required for PFV capsid assembly and that intact capsid is required for PFV reverse transcription. These data provide the first information that relates the Gag proteins of Spuma and Orthoretrovirinae and suggests a common ancestor for both lineages containing an ancient CA fold., Competing Interests: The authors have declared that no competing interests exist.

Retroviral pathogens have been an evolutionary pressure for many primate species, driving the development of an intrinsic cellular response to retroviruses and antiretroviral proteins. One such antiretroviral protein is the restriction factor Trim5α, that blocks HIV-1 infection in rhesus macaques at an early post-entry stage in the retroviral lifecycle. Trim5α self-assembles into a large hexagonal array, complimentary to the retroviral capsid. Assembly is mediated by the conserved N-terminal architecture comprising a RING domain, a Bbox domain, and a coiled coil. Recently we have shown that the Bbox domain and elements of the coiled coil form a trimer in solution, and that the Bbox domain drives assembly. During crystallisation experiments using the trimer forming construct, we determined the structure of a dimeric Bbox domain to a resolution of 1.8Å. Interface analysis reveals that residues previously shown to be required for assembly and restriction, Glu120 and Arg121, are central to the interface. Comparison to a mutant Trim5α dimer interface shows a translation of the Bbox dimerisation interface removing interactions important in the wildtype protein., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Peroxiredoxins are antioxidant proteins primarily responsible for detoxification of hydroperoxides in cells. On exposure to various cellular stresses, peroxiredoxins can acquire chaperone activity, manifested as quaternary reorganization into a high molecular weight (HMW) form. Acidification, for example, causes dodecameric rings of human peroxiredoxin 3 (HsPrx3) to stack into long helical filaments. In this work, a 4.1-Å resolution structure of low-pH-instigated helical filaments was elucidated, showing a locally unfolded active site and partially folded C terminus. A 2.8-Å crystal structure of HsPrx3 was determined at pH 8.5 under reducing conditions, wherein dodecameric rings are arranged as a short stack, with symmetry similar to low-pH filaments. In contrast to previous observations, the crystal structure displays both a fully folded active site and ordered C terminus, suggesting that the HsPrx3 HMW form maintains catalytic activity. We propose a new role for the HMW form as a self-chaperoning assembly maintaining HsPrx3 function under stress., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The post-entry restriction factor Trim5α blocks infection of retroviral pathogens shortly after the virus gains entry to the cell, preventing reverse transcription and integration into the host genome. Central to the mechanism of restriction is recognition of the lattice of capsid protein that forms the inner-shell of the retrovirus. To recognise this lattice, Trim5α has been shown to assemble into a large hexagonal array, complementary to the capsid lattice. Structures of the Trim5α coiled-coil region reveal an elongated anti-parallel dimer consistent with the edges of this array placing the Bbox domain at each end of the coiled-coil to facilitate assembly. To investigate the nature of this assembly we have designed and characterised a monomeric version of the TRIM RBCC motif with a truncated coiled-coil. Biophysical characterisation by SEC-MALLS, AUC, and SAXS demonstrate that this construct forms compact folded domain that assembles into a trimer that would support the formation of a hexagonal lattice. Furthermore, the RING domain and elements of the coiled-coil region are shown to contribute to assembly. Ubiquitylation assays demonstrate that this assembly increases ubiquitylation activity providing a link from recognition of the capsid lattice and assembly to the activation of innate immune signalling and restriction.

The members of the CcdA family are integral membrane proteins that use a disulfide cascade to transport electrons from the thioredoxin-thioredoxin reductase system in the interior of the cell into the extracytoplasmic space. The core transmembrane portion of this family is often elaborated with additional hydrophilic domains that act as adapters to deliver reducing potential to targets outside the cellular membrane. To investigate the function of family members in Mycobacterium tuberculosis, the structure of the C-terminal ectodomain from Rv2874, one of three CcdA-family members present in the genome, was determined. The crystal structure, which was refined at 1.9 Å resolution with R = 0.195 and Rfree = 0.219, reveals the predicted thioredoxin-like domain with its conserved Cys-X-X-Cys active-site motif. Unexpectedly, this domain is combined with a second domain with a carbohydrate-binding module (CBM) fold, this being the first reported example of a CBM in association with a thioredoxin-like domain fold. A cavity in the CBM adjacent to the thioredoxin active site suggests a likely carbohydrate-binding site, representing a broadening of the substrate range for CcdA-family members and an expansion of the thioredoxin-domain functionality to carbohydrate modification.

The tryptophan-biosynthesis pathway is essential for Mycobacterium tuberculosis (Mtb) to cause disease, but not all of the enzymes that catalyse this pathway in this organism have been identified. The structure and function of the enzyme complex that catalyses the first committed step in the pathway, the anthranilate synthase (AS) complex, have been analysed. It is shown that the open reading frames Rv1609 (trpE) and Rv0013 (trpG) encode the chorismate-utilizing (AS-I) and glutamine amidotransferase (AS-II) subunits of the AS complex, respectively. Biochemical assays show that when these subunits are co-expressed a bifunctional AS complex is obtained. Crystallization trials on Mtb-AS unexpectedly gave crystals containing only AS-I, presumably owing to its selective crystallization from solutions containing a mixture of the AS complex and free AS-I. The three-dimensional structure reveals that Mtb-AS-I dimerizes via an interface that has not previously been seen in AS complexes. As is the case in other bacteria, it is demonstrated that Mtb-AS shows cooperative allosteric inhibition by tryptophan, which can be rationalized based on interactions at this interface. Comparative inhibition studies on Mtb-AS-I and related enzymes highlight the potential for single inhibitory compounds to target multiple chorismate-utilizing enzymes for TB drug discovery.

SAMHD1 restricts HIV-1 infection of myeloid-lineage and resting CD4+ T-cells. Most likely this occurs through deoxynucleoside triphosphate triphosphohydrolase activity that reduces cellular dNTP to a level where reverse transcriptase cannot function, although alternative mechanisms have been proposed recently. Here, we present combined structural and virological data demonstrating that in addition to allosteric activation and triphosphohydrolase activity, restriction correlates with the capacity of SAMHD1 to form "long-lived" enzymatically competent tetramers. Tetramer disruption invariably abolishes restriction but has varied effects on in vitro triphosphohydrolase activity. SAMHD1 phosphorylation also ablates restriction and tetramer formation but without affecting triphosphohydrolase steady-state kinetics. However phospho-SAMHD1 is unable to catalyse dNTP turnover under conditions of nucleotide depletion. Based on our findings we propose a model for phosphorylation-dependent regulation of SAMHD1 activity where dephosphorylation switches housekeeping SAMHD1 found in cycling cells to a high-activity stable tetrameric form that depletes and maintains low levels of dNTPs in differentiated cells.

Adiponectin, a collagenous hormone secreted abundantly from adipocytes, possesses potent antidiabetic and anti-inflammatory properties. Mediated by the conserved Cys(39) located in the variable region of the N terminus, the trimeric (low molecular weight (LMW)) adiponectin subunit assembles into different higher order complexes, e.g. hexamers (middle molecular weight (MMW)) and 12-18-mers (high molecular weight (HMW)), the latter being mostly responsible for the insulin-sensitizing activity of adiponectin. The endoplasmic reticulum (ER) chaperone ERp44 retains adiponectin in the early secretory compartment and tightly controls the oxidative state of Cys(39) and the oligomerization of adiponectin. Using cellular and in vitro assays, we show that ERp44 specifically recognizes the LMW and MMW forms but not the HMW form. Our binding assays with short peptide mimetics of adiponectin suggest that ERp44 intercepts and converts the pool of fully oxidized LMW and MMW adiponectin, but not the HMW form, into reduced trimeric precursors. These ERp44-bound precursors in the cis-Golgi may be transported back to the ER and released to enhance the population of adiponectin intermediates with appropriate oxidative state for HMW assembly, thereby underpinning the process of ERp44 quality control., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Restriction factors (RFs) form important components of host defenses to retroviral infection. The Fv1, Trim5α, and TrimCyp RFs contain N-terminal dimerization and C-terminal specificity domains that target assembled retroviral capsid (CA) proteins enclosing the viral core. However, the molecular detail of the interaction between RFs and their CA targets is unknown. Therefore, we have determined the crystal structure of the B-box and coiled-coil (BCC) region from Trim5α and used small-angle X-ray scattering to examine the solution structure of Trim5α BCC, the dimerization domain of Fv1 (Fv1Ntd), and the hybrid restriction factor Fv1Cyp comprising Fv1NtD fused to the HIV-1 binding protein Cyclophilin A (CypA). These data reveal that coiled-coil regions of Fv1 and Trim5α form extended antiparallel dimers. In Fv1Cyp, two CypA moieties are located at opposing ends, creating a molecule with a dumbbell appearance. In Trim5α, the B-boxes are located at either end of the coiled-coil, held in place by interactions with a helical motif from the L2 region of the opposing monomer. A comparative analysis of Fv1Cyp and CypA binding to a preformed HIV-1 CA lattice reveals how RF dimerization enhances the affinity of interaction through avidity effects. We conclude that the antiparallel organization of the NtD regions of Fv1 and Trim5α dimers correctly positions C-terminal specificity and N-terminal effector domains and facilitates stable binding to adjacent CA hexamers in viral cores.

Butyrivibrio proteoclasticus is a significant component of the microbial population of the rumen of dairy cattle. It is a xylan-degrading organism whose genome encodes a large number of open reading frames annotated as fiber-degrading enzymes. We have determined the three-dimensional structure of Est2A, an acetyl xylan esterase from B. proteoclasticus, at 2.1 Å resolution, along with the structure of an inactive mutant (H351A) at 2.0 Å resolution. The structure reveals two domains-a C-terminal SGNH domain and an N-terminal jelly-roll domain typical of CE2 family structures. The structures are accompanied by experimentally determined enzymatic parameters against two model substrates, para-nitrophenyl acetate and para-nitrophenyl butyrate. The suite of fiber-degrading enzymes produced by B. proteoclasticus provides a rich source of new enzymes of potential use in industrial settings., (Copyright © 2013 Wiley Periodicals, Inc.)

The Spumaretrovirinae, 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 gag genes where structurally unrelated molecules have become functionally equivalent.

Distinctive diffuse scattering in the form of diffuse rings around Bragg positions has been observed in the diffraction patterns of a crystal of the N-terminal fragment of the Gag protein from Feline Foamy Virus. It is shown that these are caused by geometric frustration as molecules try to pack on the triangular b-c mesh of the space group P6(1)22. In order to explain the strong diffuse scattering it is necessary for the crystal to contain occupational disorder such that each unit cell contains one or other of two different molecular arrangements, A and B. The frustration arises because the nearest-neighbour packing prefers neighbouring cells to be AB or BA, which cannot be achieved on all three sides of a triangle simultaneously. To explain the observation that reciprocal sections hk5n, where n = integer, contain only Bragg peaks it is necessary that A and B are identical molecular arrangements differing only by a translation of 0.2c. The implications of the disorder for solving the structure of the protein by conventional techniques as well as the possibility of using the diffuse scattering for this purpose are discussed., (© 2011 International Union of Crystallography. Printed in Singapore – all rights reserved.)

SAMHD1, an analogue of the murine interferon (IFN)-γ-induced gene Mg11 (ref. 1), has recently been identified as a human immunodeficiency virus-1 (HIV-1) restriction factor that blocks early-stage virus replication in dendritic and other myeloid cells and is the target of the lentiviral protein Vpx, which can relieve HIV-1 restriction. SAMHD1 is also associated with Aicardi-Goutières syndrome (AGS), an inflammatory encephalopathy characterized by chronic cerebrospinal fluid lymphocytosis and elevated levels of the antiviral cytokine IFN-α. The pathology associated with AGS resembles congenital viral infection, such as transplacentally acquired HIV. Here we show that human SAMHD1 is a potent dGTP-stimulated triphosphohydrolase that converts deoxynucleoside triphosphates to the constituent deoxynucleoside and inorganic triphosphate. The crystal structure of the catalytic core of SAMHD1 reveals that the protein is dimeric and indicates a molecular basis for dGTP stimulation of catalytic activity against dNTPs. We propose that SAMHD1, which is highly expressed in dendritic cells, restricts HIV-1 replication by hydrolysing the majority of cellular dNTPs, thus inhibiting reverse transcription and viral complementary DNA (cDNA) synthesis.

The restriction factor Fv1 confers resistance to murine leukemia virus (MLV), blocking progression of the viral life cycle after reverse transcription, but before integration into the host chromosome. It is known that the specificity of restriction is determined by both the restriction factor and the viral capsid (CA), but a direct interaction between Fv1 and MLV CA has not yet been demonstrated. With the development of a previously unexplored method for in vitro polymerization of MLV CA, it has now been possible to display a binding interaction between Fv1 and MLV CA. C-terminally His-tagged CA molecules were assembled on Ni-chelating lipid nanotubes, and analysis by electron microscopy revealed the formation of a regular lattice. Comparison of binding data with existing restriction data confirmed the specificity of the binding interaction, with multiple positions of both Fv1 and CA shown to influence binding specificity.

After entry into target cells, retroviruses encounter the host restriction factors such as Fv1 and TRIM5α. While it is clear that these factors target retrovirus capsid proteins (CA), recognition remains poorly defined in the absence of structural information. To better understand the binding interaction between TRIM5α and CA, we selected a panel of novel N-tropic murine leukaemia virus (N-MLV) escape mutants by a serial passage of replication competent N-MLV in rhesus macaque TRIM5α (rhTRIM5α)-positive cells using a small percentage of unrestricted cells to allow multiple rounds of virus replication. The newly identified mutations, many of which involve changes in charge, are distributed over the outer 'top' surface of N-MLV CA, including the N-terminal β-hairpin, and map up to 29 A(o) apart. Biological characterisation with a number of restriction factors revealed that only one of the new mutations affects restriction by human TRIM5α, indicating significant differences in the binding interaction between N-MLV and the two TRIM5αs, whereas three of the mutations result in dual sensitivity to Fv1(n) and Fv1(b). Structural studies of two mutants show that no major changes in the overall CA conformation are associated with escape from restriction. We conclude that interactions involving much, if not all, of the surface of CA are vital for TRIM5α binding.

Lentiviruses are widespread in a variety of vertebrates, often associated with chronic disease states. However, until the recent discovery of the prehistoric endogenous lentiviruses in rabbits (RELIK) and lemurs (PSIV), it was thought that lentiviruses had no capacity for germline integration and were only spread horizontally in an exogenous fashion. The existence of RELIK and PSIV refuted these ideas, revealing lentiviruses to be present in a range of mammals, capable of germline integration, and far more ancient than previously thought. Using Gag sequences reconstructed from the remnants of these prehistoric lentiviruses, we have produced chimeric lentiviruses capable of infecting nondividing cells and determined structures of capsid domains from PSIV and RELIK. We show that the structures from these diverse viruses are highly similar, containing features found in modern-day lentiviruses, including a functional cyclophilin-binding loop. Together, these data provide evidence for an ancient capsid-cyclophilin interaction preserved throughout lentiviral evolution., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Rna15 is a core subunit of cleavage factor IA (CFIA), an essential transcriptional 3'-end processing factor from Saccharomyces cerevisiae. CFIA is required for polyA site selection/cleavage targeting RNA sequences that surround polyadenylation sites in the 3'-UTR of RNA polymerase-II transcripts. RNA recognition by CFIA is mediated by an RNA recognition motif (RRM) contained in the Rna15 subunit of the complex. We show here that Rna15 has a strong and unexpected preference for GU containing RNAs and reveal the molecular basis for a base selectivity mechanism that accommodates G or U but discriminates against C and A bases. This mode of base selectivity is rather different to that observed in other RRM-RNA structures and is structurally conserved in CstF64, the mammalian counterpart of Rna15. Our observations provide evidence for a highly conserved mechanism of base recognition amongst the 3'-end processing complexes that interact with the U-rich or U/G-rich elements at 3'-end cleavage/polyadenylation sites.

The release of polysaccharide from the plant cell wall is a key process to release the stored energy from plant biomass. Within the ruminant digestive system, a host of commensal microorganisms speed the breakdown of plant cell matter releasing fermentable sugars. The presence of phenolic compounds, most notably ferulic acid (FA), esterified within the cell wall is thought to pose a significant impediment to the degradation of the plant cell wall. The structure of a FA esterase from the ruminant bacterium Butyrivibrio proteoclasticus has been determined in two different space groups, in both the apo-form, and the ligand bound form with FA located in the active site. The structure reveals a new lid domain that has no structural homologues in the PDB. The flexibility of the lid domain is evident by the presence of three different conformations adopted by different molecules in the crystals. In the FA-bound structures, these conformations show sequential binding and closing of the lid domain over the substrate. Enzymatic activity assays demonstrate a broad activity against plant-derived hemicellulose, releasing at least four aromatic compounds including FA, coumaric acid, coumarin-3-carboxylic acid, and cinnamic acid. The rumen is a complex ecosystem that efficiently degrades plant biomass and the genome of B. proteoclasticus contains greater than 130 enzymes, which are potentially involved in this process of which Est1E is the first to be well characterized., (2009 Wiley-Liss, Inc.)

Jaagsiekte sheep retrovirus is a betaretrovirus and the causative agent of pulmonary adenocarcinoma, a transmissible lung tumour of sheep. Here we report the crystal structure of the capsid amino-terminal domain and examine the self-association properties of Jaagsiekte sheep retrovirus capsid. We find that the structure is remarkably similar to the amino-terminal domain of the alpharetrovirus, avian leukosis virus, revealing a previously undetected evolutionary similarity. Examination of capsid self-association suggests a mode of assembly not driven by the strong capsid carboxy-terminal domain interactions that characterise capsid assembly in the lentiviruses. Based on these data, we propose this structure provides a model for the capsid of betaretroviruses including the HML-2 family of endogenous human betaretroviruses.