Petri Kursula

Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia

Amyotrophic lateral sclerosis (ALS) is a genetically heterogeneous neurodegenerative syndrome hallmarked by adult-onset loss of motor neurons. We performed exome sequencing of 252 familial ALS (fALS) and 827 control individuals. Gene-based rare variant analysis identified an exome-wide significant enrichment of eight loss-of-function (LoF) mutations in TBK1 (encoding TANK-binding kinase 1) in 13 fALS pedigrees. No enrichment of LoF mutations was observed in a targeted mutation screen of 1,010 sporadic ALS and 650 additional control individuals. Linkage analysis in four families gave an aggregate LOD score of 4.6. In vitro experiments confirmed the loss of expression of TBK1 LoF mutant alleles, or loss of interaction of the C-terminal TBK1 coiled-coil domain (CCD2) mutants with the TBK1 adaptor protein optineurin, which has been shown to be involved in ALS pathogenesis. We conclude that haploinsufficiency of TBK1 causes ALS and fronto-temporal dementia.

Recognition of a Functional Peroxisome Type 1 Target by the Dynamic Import Receptor Pex5p

Peroxisomes require the translocation of folded and functional target proteins of various sizes across the peroxisomal membrane. We have investigated the structure and function of the principal import receptor Pex5p, which recognizes targets bearing a C-terminal peroxisomal targeting signal type 1. Crystal structures of the receptor in the presence and absence of a peroxisomal target, sterol carrier protein 2, reveal major structural changes from an open, snail-like conformation into a closed, circular conformation. These changes are caused by a long loop C terminal to the 7-fold tetratricopeptide repeat segments. Mutations in residues of this loop lead to defects in peroxisomal import in human fibroblasts. The structure of the receptor/cargo complex demonstrates that the primary receptor-binding site of the cargo is structurally and topologically autonomous, enabling the cargo to retain its native structure and function.

Molecular Basis of the Death-Associated Protein Kinase–Calcium/Calmodulin Regulator Complex

The structure of the death-associated protein kinase and calmodulin complex reveals how calmodulin binding leads to kinase activation. The Long and Short of DAPK Binding Calcium-bound calmodulin (CaM) binds to and activates CaM-dependent protein kinases (CaMKs); however, the precise mechanism whereby CaM binding activates CaMKs has been unclear. Here, de Diego et al. describe the crystal structure of a prototypical CaMK, death-associated protein kinase (DAPK), in a complex with CaM to determine how CaM binding regulates DAPK activity. Intriguingly, they found that the conformation of CaM, when in a complex with DAPK protein, was distinct from the conformation that it assumed when in a complex with a short CaM-binding peptide (the DAPK autoregulatory domain). Death-associated protein kinase (DAPK) provides a model for calcium-bound calmodulin (CaM)–dependent protein kinases (CaMKs). Here, we report the crystal structure of the binary DAPK-CaM complex, using a construct that includes the DAPK catalytic domain and adjacent autoregulatory domain. When DAPK was in a complex with CaM, the DAPK autoregulatory domain formed a long seven-turn helix. This DAPK-CaM module interacted with the DAPK catalytic domain through two separate domain-domain interfaces, which involved the upper and the lower lobe of the catalytic domain. When bound to DAPK, CaM adopted an extended conformation, which was different from that in CaM-CaMK peptide complexes. Complementary biochemical analysis showed that the ability of DAPK to bind CaM correlated with its catalytic activity. Because many features of CaM binding are conserved in other CaMKs, our findings likely provide a generally applicable model for regulation of CaMK activity.

Accurate solution structures of proteins from X-ray data and a minimal set of NMR data: calmodulin-peptide complexes as examples.

A strategy for the accurate determination of protein solution structures starting from X-ray data and a minimal set of NMR data is proposed and successfully applied to two complexes of calmodulin (CaM) with target peptides not previously described. Its implementation in the present case is based on the use of lanthanide ions as substitutes for calcium in one of the four calcium binding sites of CaM and the collection of pseudocontact shift (pcs) and residual dipolar coupling (rdc) restraints induced by the paramagnetic metals. Starting from the crystal structures, new structural models are calculated that are in excellent agreement with the paramagnetic restraints and differ significantly from the starting crystal structures. In particular, in both complexes, a change in orientation of the first helix of the N-terminal CaM domain and of the whole C-terminal domain is observed. The simultaneous use of paramagnetic pcs and rdc restraints has the following crucial advantages: (i) it allows one to assess the possible presence of interdomain conformational freedom, which cannot be detected if the rdc values are derived from external orienting media; (ii) in the absence of significant conformational freedom, the global orientation tensor can be independently and precisely determined from pcs values, which are less sensitive than rdc values to the presence of local structural inaccuracies, and therefore (iii) the relative rearrangement of a domain or a secondary structure element with respect to the metal-bearing domain can be detected.

The structure of human collapsin response mediator protein 2, a regulator of axonal growth.

Axonal growth cone guidance is a central process in nervous system development and repair. Collapsin response mediator protein 2 (CRMP‐2) is a neurite extension‐promoting neuronal cytosolic molecule involved in the signalling of growth inhibitory cues from external stimuli, such as semaphorin 3A and the myelin‐associated glycoprotein. We have determined the crystal structure of human tetrameric CRMP‐2, which is structurally related to the dihydropyriminidases; however, the active site is not conserved. The wealth of earlier functional mapping data for CRMP‐2 are discussed in light of the three‐dimensional structure of the protein. The differences in oligomerisation interfaces between CRMP‐1 and CRMP‐2 are used to model CRMP‐1/2 heterotetramers.

Structural properties of proteins specific to the myelin sheath

Summary.The myelin sheath is an insulating membrane layer surrounding myelinated axons in vertebrates, which is formed when the plasma membrane of an oligodendrocyte or a Schwann cell wraps itself around the axon. A large fraction of the total protein in this membrane layer is comprised of only a small number of individual proteins, which have certain intriguing structural properties. The myelin proteins are implicated in a number of neurological diseases, including, for example, autoimmune diseases and peripheral neuropathies. In this review, the structural properties of a number of myelin-specific proteins are described.

Structural Basis for Parasite-Specific Functions of the Divergent Profilin of Plasmodium falciparum

Profilins are key regulators of actin dynamics. They sequester actin monomers, forming a pool for rapid polymer formation stimulated by proteins such as formins. Apicomplexan parasites utilize a highly specialized microfilament system for motility and host cell invasion. Their genomes encode only a small number of divergent actin regulators. We present the first crystal structure of an apicomplexan profilin, that of the malaria parasite Plasmodium falciparum, alone and in complex with a polyproline ligand peptide. The most striking feature of Plasmodium profilin is a unique minidomain consisting of a large beta-hairpin extension common to all apicomplexan parasites, and an acidic loop specific for Plasmodium species. Reverse genetics in the rodent malaria model, Plasmodium berghei, suggests that profilin is essential for the invasive blood stages of the parasite. Together, our data establish the structural basis for understanding the functions of profilin in the malaria parasite.

Interaction between the C-terminal region of human myelin basic protein and calmodulin: analysis of complex formation and solution structure.

BackgroundThe myelin sheath is a multilamellar membrane structure wrapped around the axon, enabling the saltatory conduction of nerve impulses in vertebrates. Myelin basic protein, one of the most abundant myelin-specific proteins, is an intrinsically disordered protein that has been shown to bind calmodulin. In this study, we focus on a 19-mer synthetic peptide from the predicted calmodulin-binding segment near the C-terminus of human myelin basic protein.ResultsThe interaction of native human myelin basic protein with calmodulin was confirmed by affinity chromatography. The binding of the myelin basic protein peptide to calmodulin was tested with isothermal titration calorimetry (ITC) in different temperatures, and Kd was observed to be in the low μM range, as previously observed for full-length myelin basic protein. Surface plasmon resonance showed that the peptide bound to calmodulin, and binding was accompanied by a conformational change; furthermore, gel filtration chromatography indicated a decrease in the hydrodynamic radius of calmodulin in the presence of the peptide. NMR spectroscopy was used to map the binding area to reside mainly within the hydrophobic pocket of the C-terminal lobe of calmodulin. The solution structure obtained by small-angle X-ray scattering indicates binding of the myelin basic protein peptide into the interlobal groove of calmodulin, while calmodulin remains in an extended conformation.ConclusionTaken together, our results give a detailed structural insight into the interaction of calmodulin with a C-terminal segment of a major myelin protein, the myelin basic protein. The used 19-mer peptide interacts mainly with the C-terminal lobe of calmodulin, and a conformational change accompanies binding, suggesting a novel mode of calmodulin-target protein interaction. Calmodulin does not collapse and wrap around the peptide tightly; instead, it remains in an extended conformation in the solution structure. The observed affinity can be physiologically relevant, given the high abundance of both binding partners in the nervous system.

XDSi: a graphical interface for the data processing program XDS

With the ever-increasing automation of protein structure solution by X-ray crystallography, data collection and processing are the last experimental steps that can affect the quality of the data and, thus, the quality of the eventual model. For several dif®cult cases, such as high crystal mosaicity, the data processing program XDS (Kabsch, 1993) has proven to perform exceptionally well. However, especially for beginners, such as students learning both crystallography and the Unix system for the ®rst time, the use of the program has often been dif®cult. All user input is written to a single detector-speci®c input ®le, which ®rst has to be retrieved from a separate directory, using a text editor, and the program is started from the command line. In addition, in order to view the output, shell windows or text editors have to be used. In problematic cases, successful processing generally requires a number of runs, with editing input and examining output for each cycle. This can be frustrating when one has to run the program over and over again in a dif®cult case. Furthermore, the conversion of the XDS output re ̄ection ®les into a format recognized by the popular crystallography package CCP4 (Collaborative Computational Project, Number 4, 1994) is not straightforward.

A Structural Insight Into Lead Neurotoxicity and Calmodulin Activation by Heavy Metals.

Calmodulin is a calcium sensor that is also capable of binding and being activated by other metal ions. Of specific interest in this respect is lead, which is known to be neurotoxic and to have a very high affinity towards calmodulin. Crystal structures of human calmodulin complexed with lead and barium ions have been solved. The results will help in understanding the activation mechanisms of calmodulin by different heavy metals and will provide a detailed view of a putative target for lead neurotoxicity in humans.