Изучение белок-белковых взаимодействий лежит в основе расшифровки практически любого клеточного процесса. Важность картирования взаимодействий также определяется их ролью в качестве мишени для разнообразных лекарственных препаратов. На сегодняшний день разработано множество методов для установления самого факта взаимодействия, однако определение зоны непосредственного контакта между двумя или более взаимодействующими белковыми молекулами все еще представляет значительную сложность. Хотя такие зоны обычно представлены достаточно консервативными аминокислотными остатками, расположение интерфейса взаимодействия внутри белкового комплекса сильно затрудняет их идентификацию.

Авторы работы, недавно опубликованной в Nature Communications, предложили новый эффективный метод идентификации зон молекулярного контакта, основанный на использовании распространенных низкомолекулярных красителей (органических пигментов). Принцип метода довольно прост – добавляемое в раствор взаимодействующих белков вещество связывается со всей поверхностью белковых молекул, исключая зону непосредственного межмолекулярного взаимодействия. В результате на поверхности белка образуется защитная пленка красителя. Затем исследуемые белки подвергают трипсинолизу. При этом связавшийся краситель блокирует сайты связывания трипсина и препятствует расщеплению той части белка, которая непосредственно не участвует во взаимодействии. Триптические пептиды, соответствующие зоне межмолекулярного контакта, затем идентифицируются с помощью масс-спектрометрии.

Авторы исследования продемонстрировали работоспособность предложенного метода на примере тройного взаимодействия интерлейкина IL1β, его рецептора IL1RI и вспомогательного белка IL1RAcP. Полученные в эксперименте данные были затем использованы для создания пептидов и моноклональных антител, специфически блокирующих рассматриваемые белок-белковые взаимодействия и соответствующий интерлейкин-опосредованный сигнальный каскад. Авторы считают, что предлагаемый метод идентификации зон взаимодействия достаточно универсален и может быть использован для разработки терапевтических средств, основанных на специфической блокаде белок-белковых взаимодействий. Следует отметить, что метод потенциально может быть модифицирован для изучения взаимодействий белковых молекул с небелковыми веществами, например, с нуклеиновыми кислотами и низкомолекулярными соединениями.


Источник: Luchini A, Espina V, Liotta LA. Protein painting reveals solvent-excluded drug targets hidden within native protein-protein interfaces. Nat Commun. 2014. 5:4413.

Статья в свободном доступе.

Ссылка на патентную заявку с описанием использовавшихся красителей: http://www.google.com/patents/WO2014113433A1?cl=en

Подпись к рисунку из обсуждаемой статьи: Принцип предлагаемого метода.
(a) Молекулы красителя связываются с поверхностью белкового комплекса, но не могут попасть в недоступную для растворителя зону межмолекулярного контакта. Приведено изображение лиганд-рецепторного комплекса интерлейкина 1b со связавшимся красителем в соответствии с масштабом.
(b) Молекулы красителя, нековалентно связавшиеся рядом с консенсусными сайтами узнавания трипсина, блокируют трипсинолиз. После диссоциации окрашенных белков зона взаимодействия остается неокрашенной и подвержена трипсинолизу. Таким образом, триптические петиды будут происходить исключительно из неокрашенной зоны взаимодействия.

авторский текст

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