Structural and functional analysis of cystatin E reveals enzymologically relevant dimer and amyloid fibril states.

Protein activity is often regulated by altering the oligomerization state. One mechanism of multimerization involves domain swapping, wherein proteins exchange parts of their structures and thereby form long-lived dimers or multimers. Domain swapping has been specifically observed in amyloidogenic proteins, for example the cystatin superfamily of cysteine protease inhibitors. Cystatins are twin-headed inhibitors, simultaneously targeting the lysosomal cathepsins and legumain, with important roles in cancer progression and Alzheimer's disease. Although cystatin E is the most potent legumain inhibitor identified so far, nothing is known about its propensity to oligomerize. In this study, we show that conformational destabilization of cystatin E leads to the formation of a domain-swapped dimer with increased conformational stability. This dimer was active as a legumain inhibitor by forming a trimeric complex. By contrast, the binding sites toward papain-like proteases were buried within the cystatin E dimer. We also showed that the dimers could further convert to amyloid fibrils. Unexpectedly, cystatin E amyloid fibrils contained functional protein, which inhibited both legumain and papain-like enzymes. Fibril formation was further regulated by glycosylation. We speculate that cystatin amyloid fibrils might serve as a binding platform to stabilize the pH-sensitive legumain and cathepsins in the extracellular environment, contributing to their physiological and pathological functions.

Dissecting FOXP2 Oligomerization and DNA Binding.

Protein-protein and protein-substrate interactions are critical to function and often depend on factors that are difficult to disentangle. Herein, a combined biochemical and biophysical approach, based on electrically switchable DNA biochips and single-molecule mass analysis, was used to characterize the DNA binding and protein oligomerization of the transcription factor, forkhead box protein P2 (FOXP2). FOXP2 contains domains commonly involved in nucleic-acid binding and protein oligomerization, such as a C2 H2 -zinc finger (ZF), and a leucine zipper (LZ), whose roles in FOXP2 remain largely unknown. We found that the LZ mediates FOXP2 dimerization via coiled-coil formation but also contributes to DNA binding. The ZF contributes to protein dimerization when the LZ coiled-coil is intact, but it is not involved in DNA binding. The forkhead domain (FHD) is the key driver of DNA binding. Our data contributes to understanding the mechanisms behind the transcriptional activity of FOXP2.

Headless C1q: a new molecular tool to decipher its collagen-like functions.

Complement component C1q, a soluble defense collagen, is the recognition protein of the classical complement pathway. C1q is able to recognize and interact with multiple targets and, via the subsequent activation of its cognate serine proteases C1r and C1s, initiates the complement cascade. C1q is made up of six ABC heterotrimers each containing two different functional regions, an N-terminal collagen-like region (CLR) and a C-terminal globular region (GR). These heterotrimers assemble via their N-terminal regions, resulting in the characteristic 'bouquet-like' shape of C1q with an N-terminal bundle of collagen fibers with six diverging stems each exhibiting a C-terminal globular head. The GRs are responsible for the versatile recognition of multiple C1q targets, whereas the CLRs trigger immune response through interacting with several cellular or soluble partners. We report here the generation of the first recombinant form of human C1q without its recognition globular heads. The noncollagenous domain 2 (nc2) of type IX collagen has been substituted for the C1q GR in order to control the correct registering of the collagen triple helices of C1q chains A, B, and C. The resulting CLR_nc2 recombinant protein produced in stably transfected EXPI293 mammalian cells was correctly assembled and folded, as demonstrated by mass spectrometry, mass photometry, and electron microscopy experiments. Its interaction properties were investigated using surface plasmon resonance analysis with known CLR ligands: the tetramer of C1r and C1s dimers and MBL-associated protein MAp44. Comparison with the interaction properties of native serum-derived C1q and CLR revealed that recombinant CLR_nc2 retains C1q CLR functional properties.