Dennis Madsen

Evaluation of protein fold comparison servers

When a new protein structure has been determined, comparison with the database of known structures enables classification of its fold as new or belonging to a known class of proteins. This in turn may provide clues about the function of the protein. A large number of fold comparison programs have been developed, but they have never been subjected to a comprehensive and critical comparative analysis. Here we describe an evaluation of 11 publicly available, Web‐based servers for automatic fold comparison. Both their functionality (e.g., user interface, presentation, and annotation of results) and their performance (i.e., how well established structural similarities are recognized) were assessed. The servers were subjected to a battery of performance tests covering a broad spectrum of folds as well as special cases, such as multidomain proteins, Cα‐only models, new folds, and NMR‐based models. The CATH structural classification system was used as a reference. These tests revealed the strong and weak sides of each server. On the whole, CE, DALI, MATRAS, and VAST showed the best performance, but none of the servers achieved a 100% success rate. Where no structurally similar proteins are found by any individual server, it is recommended to try one or two other servers before any conclusions concerning the novelty of a fold are put on paper. Proteins 2004.

Interactive motif and fold recognition in protein structures

Two previously developed software tools for pattern recognition in protein structures at the level of the fold (DEJAVU) and at the level of assemblies of individual residues (SPASM) have been improved in order to increase their specificity significantly. In addition, web-servers have been developed for both tools thereby greatly enhancing the ease with which even non-experts can employ them.

New crystal structures of human glutathione transferase A1-1 shed light on glutathione binding and the conformation of the C-terminal helix.

Human glutathione transferase A1-1 is a well studied enzyme, but despite a wealth of structural and biochemical data a number of aspects of its catalytic function are still poorly understood. Here, five new crystal structures of this enzyme are described that provide several insights. Firstly, the structure of a complex of the wild-type human enzyme with glutathione was determined for the first time at 2.0 angstroms resolution. This reveals that glutathione binds in the G site in a very similar fashion as the glutathione portion of substrate analogues in other structures and also that glutathione binding alone is sufficient to stabilize the C-terminal helix of the protein. Secondly, we have studied the complex with a decarboxylated glutathione conjugate that is known to dramatically decrease the activity of the enzyme. The T68E mutant of human glutathione transferase A1-1 recovers some of the activity that is lost with the decarboxylated glutathione, but our structures of this mutant show that none of the earlier explanations of this phenomenon are likely to be correct. Thirdly, and serendipitously, the apo structures also reveal the conformation of the crucial C-terminal region that is disordered in all previous apo structures. The C-terminal region can adopt an ordered helix-like structure even in the apo state, but shows a strong tendency to unwind. Different conformations of the C-terminal regions were observed in the apo states of the two monomers, which suggests that cooperativity could play a role in the activity of the enzyme.

GFRAL is the receptor for GDF15 and is required for the anti-obesity effects of the ligand.

Growth differentiation factor 15 (GDF15; also known as MIC-1) is a divergent member of the TGF-β superfamily and is associated with body-weight regulation in humans and rodents. However, the cognate receptor of GDF15 is unknown. Here we show that GDF15 binds specifically to GDNF family receptor α-like (GFRAL) with high affinity, and that GFRAL requires association with the coreceptor RET to elicit intracellular signaling in response to GDF15 stimulation. We also found that GDF15-mediated reductions in food intake and body weight of mice with obesity were abolished in GFRAL-knockout mice. We further found that GFRAL expression was limited to hindbrain neurons and not present in peripheral tissues, which suggests that GDF15–GFRAL-mediated regulation of food intake is by a central mechanism. Lastly, given that GDF15 did not increase energy expenditure in treated mice with obesity, the anti-obesity actions of the cytokine are likely driven primarily by a reduction in food intake.

The existence of multiple conformers of interleukin-21 directs engineering of a superpotent analogue.

The high resolution three-dimensional structure of human interleukin (hIL)-21 has been resolved by heteronuclear NMR spectroscopy. Overall, the hIL-21 structure is dominated by a well defined central four-helical bundle, arranged in an up-up-down-down topology, as observed for other cytokines. A segment of the hIL-21 molecule that includes the third helical segment, helix C, is observed to exist in two distinct and interchangeable states. In one conformer, the helix C segment is presented in a regular, α-helical conformation, whereas in the other conformer, this segment is largely disordered. A structure-based sequence alignment of hIL-21 with receptor complexes of the related cytokines, interleukin-2 and -4, implied that this particular segment is involved in receptor binding. An hIL-21 analog was designed to stabilize the region around helix C through the introduction of a segment grafted from hIL-4. This novel hIL-21 analog was demonstrated to exhibit a 10-fold increase in potency in a cellular assay.

Construction of a versatile expression library for all human single-pass transmembrane proteins for receptor pairings by high throughput screening.

Interactions between protein ligands and receptors play crucial roles in cell-cell signalling. Most of the human cell surface receptors have been identified in the post-Human Genome Project era but many of their corresponding ligands remain unknown. To facilitate the pairing of orphan receptors, 2762 sequences encoding all human single-pass transmembrane proteins were selected for inclusion into a mammalian-cell expression library. This expression library, consisting of all the individual extracellular domains (ECDs), was constructed as a Fab fusion for each protein. In this format, individual ECD can be produced as a soluble protein or displayed on cell surface, depending on the applied heavy-chain Fab configuration. The unique design of the Fab fusion concept used in the library led to not only superior success rate of protein production, but also versatile applications in various high-throughput screening paradigms including protein-protein binding assays as well as cell binding assays, which were not possible for any other existing expression libraries. The protein library was screened against human coagulation factor VIIa (FVIIa), an approved therapeutic for the treatment of hemophilia, for binding partners by AlphaScreen and ForteBio assays. Two previously known physiological ligands of FVIIa, tissue factor (TF) and endothelial protein C receptor (EPCR) were identified by both assays. The cell surface displayed library was screened against V-domain Ig suppressor of T-cell activation (VISTA), an important immune-checkpoint regulator. Immunoglobulin superfamily member 11 (IgSF11), a potential target for cancer immunotherapy, was identified as a new and previously undescribed binding partner for VISTA. The specificity of the binding was confirmed and validated by both fluorescence-activated cell sorting (FACS) and surface plasmon resonance (SPR) assays in different experimental setups.