Ida Johansson

In situ proteolysis for protein crystallization and structure determination

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.

The Crystal Structure of the Human Toll-like Receptor 10 Cytoplasmic Domain Reveals a Putative Signaling Dimer

The Toll/interleukin-1 receptor (TIR) domain is a highly conserved signaling domain found in the intracellular regions of Toll-like receptors (TLRs), in interleukin-1 receptors, and in several cytoplasmic adaptor proteins. TIR domains mediate receptor signal transduction through recruitment of adaptor proteins and play critical roles in the innate immune response and inflammation. This work presents the 2.2Å crystal structure of the TIR domain of human TLR10, revealing a symmetric dimer in the asymmetric unit. The dimer interaction surface contains residues from the BB-loop, DD-loop, and αC-helix, which have previously been identified as important structural motifs for signaling in homologous TLR receptors. The interaction surface is extensive, containing a central hydrophobic patch surrounded by polar residues. The BB-loop forms a tight interaction, where a range of consecutive residues binds in a pocket formed by the reciprocal BB-loop and αC-helix. This pocket appears to be well suited for binding peptide substrates, which is consistent with the notion that peptides and peptide mimetics of the BB-loop are inhibitors for TLR signaling. The TLR10 structure is in good agreement with available biochemical data on TLR receptors and is likely to provide a good model for the physiological dimer.

The use of systematic N- and C-terminal deletions to promote production and structural studies of recombinant proteins.

Bacterial over-expression of proteins is a powerful tool to obtain soluble protein amenable to biochemical, biophysical and/or structural characterization. However, it is well established that many recombinant proteins cannot be produced in a soluble form. Several theoretical and empirical methods to improve soluble production have been suggested, although there is to date no universally accepted protocol. This report describes, and quantitatively analyses, a systematic multi-construct approach to obtain soluble protein. Although commonly used in several laboratories, quantitative analyses of the merits of the strategy applied to a larger number of target proteins are missing from the literature. In this study, typically 10 different protein constructs were tested for each targeted domain of nearly 400 human proteins. Overall, soluble expression was obtained for nearly 50% of the human target proteins upon over-expression in Escherichia coli. The chance of obtaining soluble expression was almost doubled using the multi-construct method as compared to more traditional approaches. Soluble protein constructs were subsequently subjected to crystallization trials and the multi-construct approach yielded a more than fourfold increase, from 15 proteins to 65, for the likelihood of obtaining well-diffracting crystals. The results also demonstrate the value of testing multiple constructs in crystallization trials. Finally, a retrospective analysis of gel filtration profiles indicates that these could be used with caution to prioritize protein targets for crystallization trials.

Structural basis for the interaction between tankyrase-2 and a potent Wnt-signaling inhibitor.

We report two crystal structures of the PARP domain of human tankyrase-2 (TNKS2). Tankyrases are involved in fundamental cellular processes such as telomere homeostasis and Wnt signaling. The complex of TNKS2 with the potent inhibitor XAV939 provides insights into the molecular basis of the strong interaction and suggests routes for further development of tankyrase inhibitors.

Enabling IMAC purification of low abundance recombinant proteins from E. coli lysates.

To the Editor: Currently, the most widely used method for purifying recombinant proteins for biochemical and especially structural studies is immobilized metal affinity chromatography (IMAC), in which a metal-binding polyhistidine tag (His tag) serves as a small purification handle on the target protein. IMAC is a powerful and generic purification method, with high recovery yields and low costs. Additionally, the His tag is compatible with most downstream applications because it is small and relatively inert1,2. Escherichia coli is by far the most popular expression host owing to its supremacy regarding cost, biomass production and technical simplicity3,4. However, a serious drawback of IMAC is the often-experienced failure to purify low-abundance His-tagged proteins from E. coli lysates; increasing the culture size and thereby increasing the amount of available Histagged protein does not result in increased yield. We examined this issue and propose that it is tightly linked to metal-ion leakage from the columns induced by the E. coli lysate. We used His-tagged GFP (His6-GFP) to examine the effect of E. coli lysate on the protein binding capacity of IMAC columns. Application of the soluble fraction of E. coli lysate lacking recombinant protein expression to a 1 ml HiTrap Chelating HP column (GE Healthcare) partly loaded with His6-GFP, caused extensive migration of His6-GFP whereas application of wash buffer did not (Supplementary Fig. 1a).We confirmed this using different column materials and concluded that E. coli lysate severely reduces the binding capacity of the column (data not shown). By separating a lysate into highand low-molecular-weight components we found that the reduced binding capacity was brought about by low-molecularweight components, and not high-molecular-weight components (Supplementary Fig. 1b), implying that the underlying cause for the reduced target protein binding is not the result of native E. coli proteins competing with the His-tagged protein for the immobilized nickel-ion binding sites. We determined the amount of nickel present on the different columns before and after sample load and found that the decrease in binding capacity correlated with loss of immobilized nickel ions from the column (Supplementary Fig. 1c). IMAC is very sensitive to the presence of metal chelators1, and the E. coli lysate contains many unspecific weak chelators such as dicarboxylic acids from the citric acid cycle. Under stress conditions, E. coli can also produce highly specific metal chelators, metallophores5. We speculated that such metallophores, if produced, would be mainly associated with the periplasmic space of E. coli but not with the cytosol. We therefore hypothesized that removing the periplasmic material before cell lysis could improve His-tagged recombinant protein purification yields. We subjected E. coli cells to osmotic shock to remove the periplasmic material before cell lysis (Supplementary Methods). His6-GFP did not migrate substantially on IMAC columns treated with lysate devoid of periplasmic the core signaling pathway. RNAiCut was robust to Z-score noise generated by randomly scrambling close Z scores (Supplementary Fig. 11 and Supplementary Table 5). We offer an online server (http://rnaicut.csail.mit.edu) for interpreting functional genomic experiments. Although we developed RNAiCut using a fly PPI network, RNAiCut can also be run on nonfly and non-PPI networks (Supplementary Fig. 12). This tool will help functional genomics research by enabling hit-list gene selection using orthogonal datasets.

Crystal Structure of the ATPase Domain of the Human AAA+ Protein Paraplegin/SPG7

Paraplegin is an m-AAA protease of the mitochondrial inner membrane that is linked to hereditary spastic paraplegias. The gene encodes an FtsH-homology protease domain in tandem with an AAA+ homology ATPase domain. The protein is believed to form a hexamer that uses ATPase-driven conformational changes in its AAA-domain to deliver substrate peptides to its protease domain. We present the crystal structure of the AAA-domain of human paraplegin bound to ADP at 2.2 Å. This enables assignment of the roles of specific side chains within the catalytic cycle, and provides the structural basis for understanding the mechanism of disease mutations. Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

Structural determination of functional domains in early B-cell factor (EBF) family of transcription factors reveals similarities to Rel DNA-binding proteins and a novel dimerization motif.

The early B-cell factor (EBF) transcription factors are central regulators of development in several organs and tissues. This protein family shows low sequence similarity to other protein families, which is why structural information for the functional domains of these proteins is crucial to understand their biochemical features. We have used a modular approach to determine the crystal structures of the structured domains in the EBF family. The DNA binding domain reveals a striking resemblance to the DNA binding domains of the Rel homology superfamily of transcription factors but contains a unique zinc binding structure, termed zinc knuckle. Further the EBF proteins contain an IPT/TIG domain and an atypical helix-loop-helix domain with a novel type of dimerization motif. The data presented here provide insights into unique structural features of the EBF proteins and open possibilities for detailed molecular investigations of this important transcription factor family.

Structural studies of tri-functional human GART

Human purine de novo synthesis pathway contains several multi-functional enzymes, one of which, tri-functional GART, contains three enzymatic activities in a single polypeptide chain. We have solved structures of two domains bearing separate catalytic functions: glycinamide ribonucleotide synthetase and aminoimidazole ribonucleotide synthetase. Structures are compared with those of homologous enzymes from prokaryotes and analyzed in terms of the catalytic mechanism. We also report small angle X-ray scattering models for the full-length protein. These models are consistent with the enzyme forming a dimer through the middle domain. The protein has an approximate seesaw geometry where terminal enzyme units display high mobility owing to flexible linker segments. This resilient seesaw shape may facilitate internal substrate/product transfer or forwarding to other enzymes in the pathway.

Structures of Bir Domains from Human Naip and Ciap2.

The inhibitor of apoptosis (IAP) family of proteins contains key modulators of apoptosis and inflammation that interact with caspases through baculovirus IAP-repeat (BIR) domains. Overexpression of IAP proteins frequently occurs in cancer cells, thus counteracting the activated apoptotic program. The IAP proteins have therefore emerged as promising targets for cancer therapy. In this work, X-ray crystallography was used to determine the first structures of BIR domains from human NAIP and cIAP2. Both structures harbour an N-terminal tetrapeptide in the conserved peptide-binding groove. The structures reveal that these two proteins bind the tetrapeptides in a similar mode as do other BIR domains. Detailed interactions are described for the P1-P4 side chains of the peptide, providing a structural basis for peptide-specific recognition. An arginine side chain in the P3 position reveals favourable interactions with its hydrophobic moiety in the binding pocket, while hydrophobic residues in the P2 and P4 pockets make similar interactions to those seen in other BIR domain-peptide complexes. The structures also reveal how a serine in the P1 position is accommodated in the binding pockets of NAIP and cIAP2. In addition to shedding light on the specificity determinants of these two proteins, the structures should now also provide a framework for future structure-based work targeting these proteins.

The Crystal Structure of the Dachshund Domain of Human SnoN Reveals Flexibility in the Putative Protein Interaction Surface

The human SnoN is an oncoprotein that interacts with several transcription-regulatory proteins such as the histone-deacetylase, N-CoR containing co-repressor complex and Smad proteins. This study presents the crystal structure of the Dachshund homology domain of human SnoN. The structure reveals a groove composed of conserved residues with characteristic properties of a protein-interaction surface. A comparison of the 12 monomers in the asymmetric unit reveals the presence of two major conformations: an open conformation with a well accessible groove and a tight conformation with a less accessible groove. The variability in the backbone between the open and the tight conformations matches the differences seen in previously determined structures of individual Dachshund homology domains, suggesting a general plasticity within this fold family. The flexibility observed in the putative protein binding groove may enable SnoN to recognize multiple interaction partners. Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.