The Three-Fold Axis of the HIV-1 Capsid Lattice Is the Species-Specific Binding Interface for TRIM5α.

Rhesus TRIM5α (rhTRIM5α) potently restricts replication of human immunodeficiency virus type 1 (HIV-1). Restriction is mediated through direct binding of the C-terminal B30.2 domain of TRIM5α to the assembled HIV-1 capsid core. This host-pathogen interaction involves multiple capsid molecules within the hexagonal HIV-1 capsid lattice. However, the molecular details of this interaction and the precise site at which the B30.2 domain binds remain largely unknown. The human orthologue of TRIM5α (hsTRIM5α) fails to block infection by HIV-1 both in vivo and in vitro This is thought to be due to differences in binding to the capsid lattice. To map the species-specific binding surface on the HIV-1 capsid lattice, we used microscale thermophoresis and dual-focus fluorescence correlation spectroscopy to measure binding affinity of rhesus and human TRIM5α B30.2 domains to a series of HIV-1 capsid variants that mimic distinct capsid arrangements at each of the symmetry axes of the HIV-1 capsid lattice. These surrogates include previously characterized capsid oligomers, as well as a novel chemically cross-linked capsid trimer that contains cysteine substitutions near the 3-fold axis of symmetry. The results demonstrate that TRIM5α binding involves multiple capsid molecules along the 2-fold and 3-fold interfaces between hexamers and indicate that the binding interface at the 3-fold axis contributes to the well-established differences in restriction potency between TRIM5α orthologues.IMPORTANCE TRIM5α is a cellular protein that fends off infection by retroviruses through binding to the viruses' protein shell surrounding its genetic material. This shell is composed of several hundred capsid proteins arranged in a honeycomb-like hexagonal pattern that is conserved across retroviruses. By binding to the complex lattice formed by multiple capsid proteins, rather than to a single capsid monomer, TRIM5α restriction activity persists despite the high mutation rate in retroviruses such as HIV-1. In rhesus monkeys, but not in humans, TRIM5α confers resistance to HIV-1. By measuring the binding of human and rhesus TRIM5α to a series of engineered HIV-1 capsid mimics of distinct capsid lattice interfaces, we reveal the HIV-1 capsid surface critical for species-specific binding by TRIM5α.

Non-invasive in vivo imaging of tumour-associated cathepsin B by a highly selective inhibitory DARPin.

Cysteine cathepsins often contribute to cancer progression due to their overexpression in the tumour microenvironment and therefore present attractive targets for non-invasive diagnostic imaging. However, the development of highly selective and versatile small molecule probes for cathepsins has been challenging. Here, we targeted tumour-associated cathepsin B using designed ankyrin repeat proteins (DARPins). The selective DARPin 8h6 inhibited cathepsin B with picomolar affinity (Ki = 35 pM) by binding to a site with low structural conservation in cathepsins, as revealed by the X-ray structure of the complex. DARPin 8h6 blocked cathepsin B activity in tumours ex vivo and was successfully applied in in vivo optical imaging in two mouse breast cancer models, in which cathepsin B was bound to the cell membrane or secreted to the extracellular milieu by tumour and stromal cells. Our approach validates cathepsin B as a promising diagnostic and theranostic target in cancer and other inflammation-associated diseases.

NLRP3 tyrosine phosphorylation is controlled by protein tyrosine phosphatase PTPN22.

Inflammasomes form as the result of the intracellular presence of danger-associated molecular patterns and mediate the release of active IL-1ß, which influences a variety of inflammatory responses. Excessive inflammasome activation results in severe inflammatory conditions, but physiological IL-1ß secretion is necessary for intestinal homeostasis. Here, we have described a mechanism of NLRP3 inflammasome regulation by tyrosine phosphorylation of NLRP3 at Tyr861. We demonstrated that protein tyrosine phosphatase non-receptor 22 (PTPN22), variants in which are associated with chronic inflammatory disorders, dephosphorylates NLRP3 upon inflammasome induction, allowing efficient NLRP3 activation and subsequent IL-1ß release. In murine models, PTPN22 deficiency resulted in pronounced colitis, increased NLRP3 phosphorylation, but reduced levels of mature IL-1ß. Conversely, patients with inflammatory bowel disease (IBD) that carried an autoimmunity-associated PTPN22 variant had increased IL-1ß levels. Together, our results identify tyrosine phosphorylation as an important regulatory mechanism for NLRP3 that prevents aberrant inflammasome activation.

Structures of designed armadillo-repeat proteins show propagation of inter-repeat interface effects.

The armadillo repeat serves as a scaffold for the development of modular peptide-recognition modules. In order to develop such a system, three crystal structures of designed armadillo-repeat proteins with third-generation N-caps (YIII-type), four or five internal repeats (M-type) and second-generation C-caps (AII-type) were determined at 1.8 Å (His-YIIIM4AII), 2.0 Å (His-YIIIM5AII) and 1.95 Å (YIIIM5AII) resolution and compared with those of variants with third-generation C-caps. All constructs are full consensus designs in which the internal repeats have exactly the same sequence, and hence identical conformations of the internal repeats are expected. The N-cap and internal repeats M1 to M3 are indeed extremely similar, but the comparison reveals structural differences in internal repeats M4 and M5 and the C-cap. These differences are caused by long-range effects of the C-cap, contacting molecules in the crystal, and the intrinsic design of the repeat. Unfortunately, the rigid-body movement of the C-terminal part impairs the regular arrangement of internal repeats that forms the putative peptide-binding site. The second-generation C-cap improves the packing of buried residues and thereby the stability of the protein. These considerations are useful for future improvements of an armadillo-repeat-based peptide-recognition system.

Altering lamina assembly reveals lamina-dependent and -independent functions for A-type lamins.

Lamins are intermediate filament proteins that form a fibrous meshwork, called the nuclear lamina, between the inner nuclear membrane and peripheral heterochromatin of metazoan cells. The assembly and incorporation of lamin A/C into the lamina, as well as their various functions, are still not well understood. Here, we employed designed ankyrin repeat proteins (DARPins) as new experimental tools for lamin research. We screened for DARPins that specifically bound to lamin A/C, and interfered with lamin assembly in vitro and with incorporation of lamin A/C into the native lamina in living cells. The selected DARPins inhibited lamin assembly and delocalized A-type lamins to the nucleoplasm without modifying lamin expression levels or the amino acid sequence. Using these lamin binders, we demonstrate the importance of proper integration of lamin A/C into the lamina for nuclear mechanical properties and nuclear envelope integrity. Finally, our study provides evidence for cell-type-specific differences in lamin functions.

Crystal structure of TRIM20 C-terminal coiled-coil/B30.2 fragment: implications for the recognition of higher order oligomers.

Many tripartite motif-containing (TRIM) proteins, comprising RING-finger, B-Box, and coiled-coil domains, carry additional B30.2 domains on the C-terminus of the TRIM motif and are considered to be pattern recognition receptors involved in the detection of higher order oligomers (e.g. viral capsid proteins). To investigate the spatial architecture of domains in TRIM proteins we determined the crystal structure of the TRIM20Δ413 fragment at 2.4 Å resolution. This structure comprises the central helical scaffold (CHS) and C-terminal B30.2 domains and reveals an anti-parallel arrangement of CHS domains placing the B-box domains 170 Å apart from each other. Small-angle X-ray scattering confirmed that the linker between CHS and B30.2 domains is flexible in solution. The crystal structure suggests an interaction between the B30.2 domain and an extended stretch in the CHS domain, which involves residues that are mutated in the inherited disease Familial Mediterranean Fever. Dimerization of B30.2 domains by means of the CHS domain is crucial for TRIM20 to bind pro-IL-1ß in vitro. To exemplify how TRIM proteins could be involved in binding higher order oligomers we discuss three possible models for the TRIM5α/HIV-1 capsid interaction assuming different conformations of B30.2 domains.

A Transporter Motor Taken Apart: Flexibility in the Nucleotide Binding Domains of a Heterodimeric ABC Exporter.

ABC exporters are ubiquitous multidomain transport proteins that couple ATP hydrolysis at a pair of nucleotide binding domains to substrate transport across the lipid bilayer mediated by two transmembrane domains. Recently, the crystal structure of the heterodimeric ABC exporter TM287/288 was determined. One of its asymmetric ATP binding sites is called the degenerate site; it binds nucleotides tightly but is impaired in terms of ATP hydrolysis. Here we report the crystal structures of both isolated motor domains of TM287/288. Unexpectedly, structural elements constituting the degenerate ATP binding site are disordered in these crystals and become structured only in the context of the full-length transporter. In addition, hydrogen bonding patterns of key residues, including those of the catalytically important Walker B and the switch loop motifs, are fundamentally different in the solitary NBDs compared to those in the intact transport protein. The structures reveal crucial interdomain contacts that need to be established for the proper assembly of the functional transporter complex.

Specific targeting of human caspases using designed ankyrin repeat proteins.

Abstract Caspases play important roles in cell death, differentiation, and proliferation. Due to their high homology, especially of the active site, specific targeting of a particular caspase using substrate analogues is very difficult. Although commercially available small molecules based on peptides are lacking high specificity due to overlapping cleavage motives between different caspases, they are often used as specific tools. We have selected designed ankyrin repeat proteins (DARPins) against human caspases 1-9 and identified high-affinity binders for the targeted caspases, except for caspase 4. Besides previously reported caspase-specific DARPins, we generated novel DARPins (D1.73, D5.15, D6.11, D8.1, D8.4, and D9.2) and confirmed specificity for caspases 1, 5, 6, and 8 using a subset of caspase family members. In addition, we solved the crystal structure of caspase 8 in complex with DARPin D8.4. This binder interacts with non-conserved residues on the large subunit, thereby explaining its specificity. Structural analysis of this and other previously published crystal structures of caspase/DARPin complexes depicts two general binding areas either involving active site forming loops or a surface area laterally at the large subunit of the enzyme. Both surface areas involve non-conserved surface residues of caspases.

Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter.

ATP binding cassette (ABC) transporters mediate vital transport processes in every living cell. ATP hydrolysis, which fuels transport, displays positive cooperativity in numerous ABC transporters. In particular, heterodimeric ABC exporters exhibit pronounced allosteric coupling between a catalytically impaired degenerate site, where nucleotides bind tightly, and a consensus site, at which ATP is hydrolyzed in every transport cycle. Whereas the functional phenomenon of cooperativity is well described, its structural basis remains poorly understood. Here, we present the apo structure of the heterodimeric ABC exporter TM287/288 and compare it to the previously solved structure with adenosine 5'-(ß,γ-imido)triphosphate (AMP-PNP) bound at the degenerate site. In contrast to other ABC exporter structures, the nucleotide binding domains (NBDs) of TM287/288 remain in molecular contact even in the absence of nucleotides, and the arrangement of the transmembrane domains (TMDs) is not influenced by AMP-PNP binding, a notion confirmed by double electron-electron resonance (DEER) measurements. Nucleotide binding at the degenerate site results in structural rearrangements, which are transmitted to the consensus site via two D-loops located at the NBD interface. These loops owe their name from a highly conserved aspartate and are directly connected to the catalytically important Walker B motif. The D-loop at the degenerate site ties the NBDs together even in the absence of nucleotides and substitution of its aspartate by alanine is well-tolerated. By contrast, the D-loop of the consensus site is flexible and the aspartate to alanine mutation and conformational restriction by cross-linking strongly reduces ATP hydrolysis and substrate transport.

Combined inhibition of caspase 3 and caspase 7 by two highly selective DARPins slows down cellular demise.

Caspases play important roles during apoptosis, inflammation and proliferation. The high homology among family members makes selective targeting of individual caspases difficult, which is necessary to precisely define the role of these enzymes. We have selected caspase-7-specific binders from a library of DARPins (designed ankyrin repeat proteins). The DARPins D7.18 and D7.43 bind specifically to procaspase 7 and active caspase 7, but not to other members of the family. Binding of the DARPins does not affect the active enzyme, but interferes with its activation by other caspases. The crystal structure of the caspase 7-D7.18 complex elucidates the high selectivity and the mode of inhibition. Combining these caspase-7-specific DARPins with the previously reported caspase-3-inhibitory DARPin D3.4S76R reduces the activity of caspase 3 and 7 in double-transfected HeLa cells during apoptosis. In addition, these cells showed less susceptibility to TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand)-induced apoptosis in living cell experiments. D7.18 and D7.43 are therefore novel tools for in vitro studies on procaspase 7 activation as well as for clarifying the role of its activation in different cellular processes. If applied in combination with D3.4S76R, they represent an excellent instrument to increase our understanding of these enzymes during various cellular processes.

Unusual structural features revealed by the solution NMR structure of the NLRC5 caspase recruitment domain.

The cytosolic nucleotide-binding domain and leucine-rich repeat-containing receptors (NLRs) are key sensors for bacterial and viral invaders and endogenous stress signals. NLRs contain a varying N-terminal effector domain that regulates the downstream signaling events upon its activation and determines the subclass to which a NLR member belongs. NLRC5 contains an unclassified N-terminal effector domain that has been reported to interact downstream with the tandem caspase recruitment domain (CARD) of retinoic acid-inducible gene I (RIG-I). Here we report the solution structure of the N-terminal effector domain of NLRC5 and in vitro interaction experiments with the tandem CARD of RIG-I. The N-terminal effector domain of NLRC5 adopts a six α-helix bundle with a general death fold, though it displays specific structural features that are strikingly different from the CARD. Notably, α-helix 3 is replaced by an ordered loop, and α-helix 1 is devoid of the characteristic interruption. Detailed structural alignments between the N-terminal effector domains of NLRC5 with a representative of each death-fold subfamily showed that NLRC5 fits best to the CARD subfamily and can be called an atypical CARD. Due to the specific structural features, the atypical CARD also displays a different electrostatic surface. Because the shape and charge of the surface is crucial for the establishment of a homotypic CARD-CARD interaction, these specific structural features seem to have a significant effect on the interaction between the atypical CARD of NLRC5 and the tandem RIG-I CARD.

Biocrystallography in Switzerland: achievements and future perspectives.

The first protein crystallography group in Switzerland was installed at the Biozentrum of the University of Basel approximately 40 years ago. Since then protein crystallography has grown and matured remarkably and is now established in the molecular biology, biochemistry or biological medicine departments of most major Swiss Universities as well as in the pharmaceutical industry and in biotech startup companies. Swiss X-ray biocrystallography groups have made remarkable contributions from the beginning and have brought Switzerland to the forefront in biostructural research during the last 5 to 10 years. Switzerland has now a leading position in the areas of supramolecular complexes, membrane proteins and structure-based drug design in pharmaceutical and biotech industries. Protein crystallography on the outer membrane protein ompF as well as the development of the lipidic cubic phase crystallization methodology has been pioneered at the Biozentrum. The latter found its somewhat late recognition through the recent explosion in structure determinations of the seven transmembrane helix G-coupled receptors. Highlights from Swiss structural biology groups in the field of supramolecular complexes include the structures of ribosomal particles, of the nucleosome and the pilus assembly complex of uropathogenic E. coli. On the membrane protein side advances in the field of ABC transporters and ion channels are world-recognized achievements of Swiss structural biology. Dedicated laboratories at many academic and industrial institutions, their current research programs, the availability of excellent infrastructure and the continuing efforts to build new facilities such as the SwissFEL indicate an even brighter future for structural biology in Switzerland.

Biocrystallography in Switzerland: Achievements and Future Perspectives.

The first protein crystallography group in Switzerland was installed at the Biozentrum of the University of Basel approximately 40 years ago. Since then protein crystallography has grown and matured remarkably and is now established in the molecular biology, biochemistry or biological medicine departments of most major Swiss Universities as well as in the pharmaceutical industry and in biotech startup companies. Swiss X-ray biocrystallography groups have made remarkable contributions from the beginning and have brought Switzerland to the forefront in biostructural research during the last 5 to 10 years. Switzerland has now a leading position in the areas of supramolecular complexes, membrane proteins and structure-based drug design in pharmaceutical and biotech industries. Protein crystallography on the outer membrane protein ompF as well as the development of the lipidic cubic phase crystallization methodology has been pioneered at the Biozentrum. The latter found its somewhat late recognition through the recent explosion in structure determinations of the seven transmembrane helix G-coupled receptors. Highlights from Swiss structural biology groups in the field of supramolecular complexes include the structures of ribosomal particles, of the nucleosome and the pilus assembly complex of uropathogenic E. coli. On the membrane protein side advances in the field of ABC transporters and ion channels are world-recognized achievements of Swiss structural biology. Dedicated laboratories at many academic and industrial institutions, their current research programs, the availability of excellent infrastructure and the continuing efforts to build new facilities such as the SwissFEL indicate an even brighter future for structural biology in Switzerland.

Chimeric hERG channels containing a tetramerization domain are functional and stable.

Biochemical and detailed structural information of human ether-a-go-go-related gene (hERG) potassium channels are scarce but are a prerequisite to understand the unwanted interactions of hERG with drugs and the effect of mutations that lead to long QT syndrome. Despite the huge interest in hERG, to our knowledge, procedures that provide a purified, functional, and tetrameric hERG channel are not available. Here, we describe hybrid hERG molecules, termed chimeric hERG channels, in which the N-terminal Per-Arnt-Sim (PAS) domain is deleted and the C-terminal C-linker as well as the cyclic nucleotide binding domain (CNBD) portion is replaced by an artificial tetramerization domain. These chimeric hERG channels can be overexpressed in HEK cells, solubilized in detergent, and purified as tetramers. When expressed in Xenopus laevis oocytes, the chimeric channels exhibit efficient trafficking to the cell surface, whereas a hERG construct lacking the PAS and C-linker/CNBD domains is retained in the cytoplasm. The chimeric hERG channels retain essential hERG functions such as voltage-dependent gating and inhibition by astemizole and the scorpion toxin BeKm-1. The chimeric channels are thus powerful tools for helping to understand the contribution of the cytoplasmic hERG domains to the gating process and are suitable for in vitro biochemical and structural studies.

Precision is essential for efficient catalysis in an evolved Kemp eliminase.

Linus Pauling established the conceptual framework for understanding and mimicking enzymes more than six decades ago. The notion that enzymes selectively stabilize the rate-limiting transition state of the catalysed reaction relative to the bound ground state reduces the problem of design to one of molecular recognition. Nevertheless, past attempts to capitalize on this idea, for example by using transition state analogues to elicit antibodies with catalytic activities, have generally failed to deliver true enzymatic rates. The advent of computational design approaches, combined with directed evolution, has provided an opportunity to revisit this problem. Starting from a computationally designed catalyst for the Kemp elimination--a well-studied model system for proton transfer from carbon--we show that an artificial enzyme can be evolved that accelerates an elementary chemical reaction 6 × 10(8)-fold, approaching the exceptional efficiency of highly optimized natural enzymes such as triosephosphate isomerase. A 1.09 Å resolution crystal structure of the evolved enzyme indicates that familiar catalytic strategies such as shape complementarity and precisely placed catalytic groups can be successfully harnessed to afford such high rate accelerations, making us optimistic about the prospects of designing more sophisticated catalysts.

Structure and substrate-induced conformational changes of the secondary citrate/sodium symporter CitS revealed by electron crystallography.

The secondary Na+/citrate symporter CitS of Klebsiella pneumoniae is the best-characterized member of the 2-hydroxycarboxylate transporter family. The recent projection structure gave insight into its overall structural organization. Here, we present the three-dimensional map of dimeric CitS obtained with electron crystallography. Each monomer has 13 a-helical transmembrane segments; six are organized in a distal helix cluster and seven in the central dimer interface domain. Based on structural analyses and comparison to VcINDY, we propose a molecular model for CitS, assign the helices, and demonstrate the internal structural symmetry. We also present projections of CitS in several conformational states induced by the presence and absence of sodium and citrate as substrates. Citrate binding induces a defined movement of a helices within the distal helical cluster. Based on this, we propose a substrate translocation site and conformational changes that are in agreement with the transport model of ''alternating access''.

Design, construction, and characterization of a second-generation DARP in library with reduced hydrophobicity.

Designed ankyrin repeat proteins (DARPins) are well-established binding molecules based on a highly stable nonantibody scaffold. Building on 13 crystal structures of DARPin-target complexes and stability measurements of DARPin mutants, we have generated a new DARPin library containing an extended randomized surface. To counteract the enrichment of unspecific hydrophobic binders during selections against difficult targets containing hydrophobic surfaces such as membrane proteins, the frequency of apolar residues at diversified positions was drastically reduced and substituted by an increased number of tyrosines. Ribosome display selections against two human caspases and membrane transporter AcrB yielded highly enriched pools of unique and strong DARPin binders which were mainly monomeric. We noted a prominent enrichment of tryptophan residues during binder selections. A crystal structure of a representative of this library in complex with caspase-7 visualizes the key roles of both tryptophans and tyrosines in providing target contacts. These aromatic and polar side chains thus substitute the apolar residues valine, leucine, isoleucine, methionine, and phenylalanine of the original DARPins. Our work describes biophysical and structural analyses required to extend existing binder scaffolds and simplifies an existing protocol for the assembly of highly diverse synthetic binder libraries.

New concepts and aids to facilitate crystallization.

Novel tools and technologies are required to obtain structural information of difficult to crystallize complex biological systems such as membrane proteins, multiprotein assemblies, transient conformational states and intrinsically disordered proteins. One promising approach is to select a high affinity and specificity-binding partner (crystallization chaperone), form a complex with the protein of interest and crystallize the complex. Often the chaperone reduces the conformational freedom of the target protein and additionally facilitates the formation of well-ordered crystals. This review provides an update on the recent successes in chaperone-assisted crystallography. We also stress the importance of synergistic approaches involving protein engineering, crystallization chaperones and crystallization additives. Recent examples demonstrate that investment in such approaches can be key to success.