Solvent water interactions within the active site of the membrane type I matrix metalloproteinase

Matrix metalloproteinases (MMP) are an important family of proteases which catalyze the degradation of extracellular matrix components. While the mechanism of peptide cleavage is well established, the process of enzyme regeneration, which represents the rate limiting step of the catalytic cycle, remains unresolved. This step involves the loss of the newly formed N-terminus (amine) and C-terminus (carboxylate) protein fragments from the site of catalysis coupled with the inclusion of one or more solvent waters. Here we report a novel crystal structure of membrane type I MMP (MT1-MMP or MMP-14), which includes a small peptide bound at the catalytic Zn site via its C-terminus. This structure models the initial product state formed immediately after peptide cleavage but before the final proton transfer to the bound amine; the amine is not present in our system and as such proton transfer cannot occur. Modeling of the protein, including earlier structural data of Bertini and coworkers [I. Bertini, et al., Angew. Chem., Int. Ed., 2006, 45, 7952–7955], suggests that the C-terminus of the peptide is positioned to form an H-bond network to the amine site, which is mediated by a single oxygen of the functionally important Glu240 residue, facilitating efficient proton transfer. Additional quantum chemical calculations complemented with magneto-optical and magnetic resonance spectroscopies clarify the role of two additional, non-catalytic first coordination sphere waters identified in the crystal structure. One of these auxiliary waters acts to stabilize key intermediates of the reaction, while the second is proposed to facilitate C-fragment release, triggered by protonation of the amine. Together these results complete the enzymatic cycle of MMPs and provide new design criteria for inhibitors with improved efficacy.Financial support was provided by the Max Planck Gesellschaft and the Cluster of Excellence RESOLV (EXC 1069) funded by the Deutsche Forschungsgemeinschaft. I. S. is supported by the Binational Science Foundation (BSF) and Israel Science Foundation (ISF). I. S. and M. H. are also supported by the ERC Advanced Grant 695437 THz-Calorimetry. M. G. is an Awardee of the Weizmann Institute of Science National Postdoctoral Award Program for Advancing Women in Science and a recipient of an A. v. Humboldt Fellowship. N. C. acknowledges the support of the Australian Research Council: Future Fellowship (FT140100834). Open Access funding provided by the Max Planck Society

Property turnover digitalisation: Interdisciplinary problems of post-classical jurisprudence

The purpose of the research is to consider the key interdisciplinary research and practice problems of the property turnover digitalisation and propose ways to solve them. Elements, phenomena and processes of the digital environment first appeared in the countries of the Anglo-Saxon legal family. By determining the constitutive features of them, the authors turned to classical domestic and foreign legal statutory concepts and policy management. The research was carried out by the comparative-legal method and modelling method. The identification of interdisciplinary research and practice problems as well as methods for solving them was based on the methods for ascent from the abstract to the concrete, induction and deduction. As a result of the implementation of the research objectives through the general scientific and private scientific methods described above, the authors identified the constitutive features of the key elements of property turnover in the digital environment, such as digital currency, smart contract, electronic trading. Identified were both doctrinal and practical problems associated with the essence and features of those elements, as well as the legal regulation of the associated relations. Those elements are considered in two dimensions, in the information system and legal field. In addition, significant gaps in the regulatory framework of the associated relations were identified; the impossibility of applying the classical regime of objects of civil rights and the rules of documentary circulation to digital objects and processes was substantiated. The novelty of the work consists in proposing ways to solve interdisciplinary theoretical and practical problems of the property turnover digitalisation, which are important for a wide range of academic researchers and practitioners in the legal field