X-ray Structures of Human Furin in Complex with Competitive Inhibitors

Abstract

Furin inhibitors are promising therapeutics for the treatment of cancer and numerous infections caused by bacteria and viruses, including the highly lethal Bacillus anthracis or the pandemic influenza virus. Development and improvement of inhibitors for pharmacological use require a detailed knowledge of the protease’s substrate and inhibitor binding properties. Here we present a novel preparation of human furin and the first crystal structures of this enzyme in complex with noncovalent inhibitors. We show the inhibitor exchange by soaking, allowing the investigation of additional inhibitors and substrate analogues. Thus, our work provides a basis for the rational design of furin inhibitors.

Structure of the unliganded form of the proprotein convertase furin suggests activation by a substrate-induced mechanism

“…These studies revealed two activityswitches affecting the functionality of the active site dependent upon substrate and calcium, respectively. Comparing the structures of unliganded and inhibitor-bound furin, we observe a significant structural rearrangement of the substrate-binding cleft without alteration of the global fold of the protease, which provides a structural framework and should be highly conserved in orthologs of this enzyme as well (6,16,17). The active-site cleft shuttles between two structurally well-defined states of the enzyme, the inactive off and the active on states.…”

“…Details regarding the plasmids, the expression system, purification procedure, and functional analysis used for human furin were described previously (17) and are also given in SI Materials and Methods. Crystals of human furin were grown in sitting drops mixing equal volumes of the protein solution (∼9 mg/mL) and crystallization solution [100 mM Mes, 200 mM K/NaH 2 PO 4 , pH 5.5-6.0, 3-4 M NaCl, and 3% (vol/vol) DMSO].…”

“…Although Ca 2+ is not involved in the catalytic cycle per se, all PCs require calcium for their enzymatic activity (17,22,23).…”

“…Different crystal structures of the inhibitor-bound murine (16) and human (6,17) enzyme, together with structural studies of the yeast homolog Kex2p (18,19) and modeling approaches (9, 20), gave hints of how substrates and substrate-derived inhibitors bind with high specificity to furin and to the PCs, but suffer from the lack of structural information of the unliganded enzyme. The commonly accepted notion is that the PCs bind their cognate substrates via recognition at several subsites, typically involving multiple tight contacts and hydrogen bonds.…”

Furin as proprotein convertase and its role in normal and pathological biological processes

“…Рентгеноструктурный анализ комплекса показал, что фурин состоит из двух доменов: каталитического и Р-домена, которые связаны междоменным линкером и могут эффективно взаимодействовать между собой [5]. В 2014 году проведён рентгеноструктурный анализ растворимой формы фурина человека (последовательность Asp 23 -Ala 574 ) в комплексе с конкурентным ингибитором, который позволил определить координаты его активного центра и сайты, связывающие три иона Са 2+ и ион Na + , требующиеся для активности фермента, Эти исследования являются необходимым условием для создания эффективных, высокоспецифичных ингибиторов фурина нового поколения [9]. …”