Felix Simkovic

Titin kinase is an inactive pseudokinase scaffold that supports MuRF1 recruitment to the sarcomeric M-line

Striated muscle tissues undergo adaptive remodelling in response to mechanical load. This process involves the myofilament titin and, specifically, its kinase domain (TK; titin kinase) that translates mechanical signals into regulatory pathways of gene expression in the myofibril. TK mechanosensing appears mediated by a C-terminal regulatory tail (CRD) that sterically inhibits its active site. Allegedly, stretch-induced unfolding of this tail during muscle function releases TK inhibition and leads to its catalytic activation. However, the cellular pathway of TK is poorly understood and substrates proposed to date remain controversial. TKs best-established substrate is Tcap, a small structural protein of the Z-disc believed to link TK to myofibrillogenesis. Here, we show that TK is a pseudokinase with undetectable levels of catalysis and, therefore, that Tcap is not its substrate. Inactivity is the result of two atypical residues in TKs active site, M34 and E147, that do not appear compatible with canonical kinase patterns. While not mediating stretch-dependent phospho-transfers, TK binds the E3 ubiquitin ligase MuRF1 that promotes sarcomeric ubiquitination in a stress-induced manner. Given previous evidence of MuRF2 interaction, we propose that the cellular role of TK is to act as a conformationally regulated scaffold that functionally couples the ubiquitin ligases MuRF1 and MuRF2, thereby coordinating muscle-specific ubiquitination pathways and myofibril trophicity. Finally, we suggest that an evolutionary dichotomy of kinases/pseudokinases has occurred in TK-like kinases, where invertebrate members are active enzymes but vertebrate counterparts perform their signalling function as pseudokinase scaffolds.

Mechanistic and functional diversity in the mechanosensory kinases of the titin-like family

The giant cytoskeletal kinases of the titin-like family are emerging as key mediators of stretch-sensing in muscle. It is thought that their elastic conformational deformation during muscle function regulates both their catalysis and the recruitment of regulatory proteins to signalosomes that assemble in their vicinity. In the present article, we discuss the speciation of mechanosensory mechanisms in titin-like kinases, their scaffolding properties and the kinase/pseudokinase domain variations that define a rich functional diversity across the family.

Applications of contact predictions to structural biology

Recent developments allow the extraction of accurate contact predictions from multiple protein-sequence alignments. This review illustrates the manifold ways in which this information may assist the experimental structural biologist.

Residue contacts predicted by evolutionary covariance extend the application of ab initio molecular replacement to larger and more challenging protein folds

Residue-contact predictions extend the range of ab initio molecular replacement.

Potential DNA binding and nuclease functions of ComEC domains characterized in silico.

Bacterial competence, which can be natural or induced, allows the uptake of exogenous double stranded DNA (dsDNA) into a competent bacterium. This process is known as transformation. A multiprotein assembly binds and processes the dsDNA to import one strand and degrade another yet the underlying molecular mechanisms are relatively poorly understood. Here distant relationships of domains in Competence protein EC (ComEC) of Bacillus subtilis (Uniprot: P39695) were characterized. DNA‐protein interactions were investigated in silico by analyzing models for structural conservation, surface electrostatics and structure‐based DNA binding propensity; and by data‐driven macromolecular docking of DNA to models. Our findings suggest that the DUF4131 domain contains a cryptic DNA‐binding OB fold domain and that the β‐lactamase‐like domain is the hitherto cryptic competence nuclease. Proteins 2016; 84:1431–1442.

SIMBAD: a sequence-independent molecular-replacement pipeline

SIMBAD is a sequence-independent molecular-replacement pipeline for solving difficult molecular-replacement cases where contaminants have been crystallized. It can also be used to find structurally related search models where no obvious homologue can be found through sequence-based searching.

ConKit: a python interface to contact predictions

Summary: Recent advances in protein residue contact prediction algorithms have led to the emergence of many new methods and a variety of file formats. We present ConKit, an open source, modular and extensible Python interface which allows facile conversion between formats and provides an interface to analyses of sequence alignments and sets of contact predictions. Availability and Implementation: ConKit is available via the Python Package Index. The documentation can be found at http://www.conkit.org. ConKit is licensed under the BSD 3‐Clause. Contact: hlfsimko@liverpool.ac.uk or drigden@liverpool.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online.

Ensembles generated from crystal structures of single distant homologues solve challenging molecular-replacement cases in AMPLE

Novel ways to produce search models from distant homologues for molecular replacement are presented.

Approaches to ab initio molecular replacement of α-helical transmembrane proteins

Homology-independent methods for ab initio phasing of α-helical transmembrane proteins are explored.

Unconventional molecular replacement for helical transmembrane proteins using AMPLE

AMPLE is a pipeline for using unconventional models in automated MR. It has been particularly successful in its cluster-andtruncate approach to creating ensemble search models from ab initio molecular models for globular targets [1]. Recent work has focussed on exploring the viability of AMPLE’s approach to transmembrane protein targets. By sampling multiple clusters of ab initio models and using new scoring algorithms, we have been able to double the success rate of AMPLE on a difficult set of test cases without an increase in processing time. Additionally, exploring new bioinformatics approaches, such as restraining ab initio structure prediction using predicted residue-residue contacts, has pushed the boundaries of AMPLE’s success [2].