ИССЛЕДОВАНИЕ ТРАНСМЕМБРАННОГО БЕЛКА CD79B МЕТОДОМ МНОГОМЕРНОЙ ИМПУЛЬСНОЙ ЯМР СПЕКТРОСКОПИИ

In the present work, using the cell-free expression system, we prepared the proteins CD79A/CD79B labeled by stable isotopes of carbon-13 and nitrogen-15. It is shown that target proteins are mostly localized in the pellet of the cell-free expression system, which is consistent with their membrane nature and the presence of a transmembrane domain in their structure. Physicochemical parameters of the CD79A/CD79B samples were defined to obtain the multidimensional correlation NMR spectra of high resolution. The analysis of the obtained correlation spectra shows that under experimental conditions, CD79B exists in the disordered state. The splitting of the signal from the NH-group of the side chain of single tryptophan residue indicates the presence of slow conformational transitions in this region of the polypeptide chain. The addition of the trifluoroacetic acid to the solution of CD79B in DMSO leads to the destruction of the intermolecular bonds of “protein micelles” and the formation of its monomeric form that is well detectable by NMR. В настоящей работе с использованием системы бесклеточной экспрессии получены меченые стабильными изотопами углерода-13 и азота-15 белки CD79A/CD79B. Установлено, что целевые белки обнаруживаются в осадке бесклеточной системы экспрессии, что согласуется с их мембранной природой и наличием трансмембранного домена в их составе. Определены физико-химические параметры образцов CD79A/CD79B с целью получения многомерных корреляционных ЯМР спектров высокого разрешения. Анализ полученных корреляционных спектров свидетельствует, что CD79B при выбранных условиях находится в неупорядоченном состоянии. Расщепление сигнала от NH- группы боковой цепи единственного остатка триптофана указывает на наличие медленных конформационных превращений в этой области полипептидной цепи. Добавление трифторуксусной кислоты к раствору CD79B в диметил- сульфоксиде приводит к разрушению межмолекулярных связей «белковой мицеллы» и образованию его мономерной формы, хорошо детектируемой ЯМР спектроскопией.

РАЗНОСТНАЯ ЯМР СПЕКТРОСКОПИЯ С ПЕРЕНОСОМ НАСЫЩЕНИЯ В ИССЛЕДОВАНИИ ВЗАИМОДЕЙСТВИЯ ЦИТОХРОМА Р450cam С 4-ФЕНИЛИМИДАЗОЛОМ: ОБНАРУЖЕНИЕ НОВОГО ПРОМЕЖУТОЧНОГО СОСТОЯНИЯ КОМПЛЕКСА

The present work is devoted to the investigation of the interaction of cytochrome P450cam with 4-phenylimidazole (4-PI) using spectrophotometry and NMR spectroscopy. The data obtained by the STD-NMR method indicate the existence of an intermediate short-lived state of 4-phenylimidazole in the active site of P450cam where 4-phenylimidazole is bound to the inner region of the active site and/or to the substrate access channel without the formation of a coordination bond between the ligand azole group atoms and the heme iron atom. In this article, we first used the STD-NMR method to study the interaction of cytochrome P450 with ligand. The equilibrium dissociation constant of the P450cam-4-PI complex, which was calculated using the dependence of the amplification factor at zero saturation time (10.4 mM), differs from the constant that was calculated at constant saturation time (34.6 mM). This fact indicates the dependence of the Kd determination using STD-NMR at saturation time and concentrations of interacting substances. Comparison of the dissociation energy for the intermediate complex (11.2 kJ) relative to the direct coordination complex (28.5 kJ) suggests that the main contribution to the protein-ligand interaction is related to the hydrophobic interaction of 4-PI with the inner surface of the cavity of the active site of P450cam. The observed intermediate state makes it possible to explain the formation of hydroxylated forms of azole inhibitors during the interaction with cytochromes P450, when an inhibitor is in an intermediate form as a substrate and is not bound by a coordination bond with a heme iron atom. Настоящая работа посвящена исследованию взаимодействия цитохрома P450cam c 4-фенилимидазолом (4-PI) методом спектрофотометрии и ЯМР спектроскопии. Полученные методом разницы переноса насыщения ЯМР (РПН-ЯМР) данные указывают на существование промежуточного короткоживущего состояния 4-фенилимидазола в активном сайте P450cam, где 4-фенилимидазол связан с внутренней областью активного сайта и/или областью канала доступа субстрата без образования координационной связи между атомами азольной группы лиганда и атомом железа гема. В данной работе нами впервые применен метод РПН-ЯМР для исследования взаимодействия цитохрома P450 c лигандом. Равновесная константа диссоциации комплекса P450cam–4-PI, рассчитанная с использованием зависимости фактора амплификации РПН при нулевом времени насыщения, составляет 10,4 мМ и отличается от константы, рассчитанной при постоянном времени насыщения (34,6 мМ), что указывает на зависимость определения Kd при использовании РПН-ЯМР от времени насыщения и концентраций взаимодействующих веществ. Сравнение энергии диссоциации для промежуточного комплекса (11,2 кДж) относительно комплекса с прямой координацией (28,5 кДж) позволяет предположить, что основной вклад во взаимодействие белок–лиганд вносит гидрофобное взаимодействие 4-PI с внутренней поверхностью полости активного сайта Р450cam. Обнаруженное промежуточное состояние позволяет объяснить образование гидроксилированных форм ингибиторов азольной природы при взаимодействии с цитохромами P450, когда ингибитор находится в промежуточной форме в качестве субстрата, без образования координационной связи с атомом железа гема.

Физико-химические свойства Δ3−12 цистеин-обедненного цитохрома P450 3A4 с аминокислотной заменой S291C

Cytochrome P450 3A4 (3A4) is highly expressed in the human liver cells and plays a decisive role in the metabolism of xenobiotics, including more than 50 % of medical products. The activity of this enzyme can be regulated at the expression level of genes, as well as at the conformation level of the structure of the protein itself, due to changes in the molecular environment, including due to the interaction with high-molecular effectors. The understanding of the structure changes and the 3A4 dynamics in response to the environmental changes is necessary to predict the changes in the level of its activity that to a considerable extent regulates the body’s homeostasis. To perform in vitro experiments on the structure, dynamics, and protein-ligand/protein interactions of the enzymes by the modern spectral methods, the approach is used, in which the target protein is selectively added with cysteine residues in the given polypeptide chain loci by the protein engineering methods for subsequent labeling with specialized molecular labels. To do this, the human mutant form of membrane-bound (full length) recombinant cytochrome P450 3A4 C58A/C64M/C98A/C239T/C377A/C468S/S291C was obtained. According to the circular dichroism spectroscopy data we established that the introduced mutations do not cause significant changes in the secondary structure of the obtained form 3A4, which shows the preservation of the folding of the peptide chain. The spectral photometric measurements were made to comparatively analyze the changes in the affinity to the ligands of the active center. Moreover, we showed that the testosterone hydroxylase activity in the in vitro reconstructed system for a given mutation form of 3A4 increases many times with respect to the wild form of the enzyme. Цитохром P450 3A4 (3A4) экспрессируется в клетках печени человека и играет ключевую роль в метаболизме ксенобиотиков, в том числе и более 50 % лекарственных препаратов. Регуляция активности данного фермента может происходить на уровне экспрессии генов, а также на уровне конформационного состояния структуры самого белка, за счет изменения молекулярного окружения, в том числе за счет взаимодействия с высокомолекулярными эффекторами. Понимание изменения структуры и динамики 3A4 в ответ на изменение условий среды необходимо для предсказания изменения уровня его активности, который в значительной степени обуславливает гомеостаз организма. Для проведения in vitro экспериментов по исследованию структуры, динамики и белоклигандных/белковых взаимодействий ферментов современными спектральными методами используется подход, в котором в целевой белок селективно вводятся методами белковой инженерии цистеиновые остатки в заданные локусы полипептидной цепи для последующего мечения специализированными молекулярными метками. Для этих целей в данной работе была получена мутантная форма мембрансвязанного (полноразмерного) рекомбинантного цитохрома P450 3A4 человека C58A/C64M/C98A/C239T/C377A/C468S/S291C. По данным спектроскопии кругового дихроизма нами было установлено, что введенные мутации не вызывают значимых изменений во вторичной структуре полученной формы 3A4, что свидетельствует о сохранении свернутости полипептидной цепи. Проведены спектрофотометрические измерения для сравнительного анализа изменения сродства к лигандам активного центра. Более того, нами было показано, что тестостерон гидроксилирующая активность в in vitro реконструированной системе для данной мутантной формы 3A4 многократно увеличивается относительно дикой формы фермента

Elucidation of the Conformational Transition of Oligopeptidase B by an Integrative Approach Based on the Combination of X-ray, SAXS, and Essential Dynamics Sampling Simulation

Oligopeptidase B (OPB) is the least studied group from the prolyl oligopeptidase family. OPBs are found in bacteria and parasitic protozoa and represent pathogenesis factors of the corresponding infections. OPBs consist of two domains connected by a hinge region and have the characteristics of conformational dynamics, which include two types of movements: the bridging/separation of α/β-hydrolase catalytic and β-propeller-regulatory domains and the movement of a loop carrying catalytic histidine, which regulates an assembly/disassembly of the catalytic triad. In this work, an elucidation of the interdomain dynamics of OPB from Serratia proteamaculans (SpOPB) with and without modification of the hinge region was performed using a combination of X-ray diffraction analysis and small-angle X-ray scattering, which was complemented with an essential dynamics sampling (EDS) simulation. The first crystal structure of catalytically deficient SpOPB (SpOPBS532A) with an intact hinge sequence is reported. Similarly to SpOPB with modified hinges, SpOPBS532A was crystallized in the presence of spermine and adopted an intermediate conformation in the crystal lattice. Despite the similarity of the crystal structures, a difference in the catalytic triad residue arrangement was detected, which explained the inhibitory effect of the hinge modification. The SpOPBS532A structure reconstituted to the wild-type form was used as a starting point to the classical MD followed by EDS simulation, which allowed us to simulate the domain separation and the transition of the enzyme from the intermediate to open conformation. The obtained open state model was in good agreement with the experimental SAXS data

Elucidation of the Conformational Transition of Oligopeptidase B by an Integrative Approach Based on the Combination of X-ray, SAXS, and Essential Dynamics Sampling Simulation

Oligopeptidase B (OPB) is the least studied group from the prolyl oligopeptidase family. OPBs are found in bacteria and parasitic protozoa and represent pathogenesis factors of the corresponding infections. OPBs consist of two domains connected by a hinge region and have the characteristics of conformational dynamics, which include two types of movements: the bridging/separation of α/β-hydrolase catalytic and β-propeller-regulatory domains and the movement of a loop carrying catalytic histidine, which regulates an assembly/disassembly of the catalytic triad. In this work, an elucidation of the interdomain dynamics of OPB from Serratia proteamaculans (SpOPB) with and without modification of the hinge region was performed using a combination of X-ray diffraction analysis and small-angle X-ray scattering, which was complemented with an essential dynamics sampling (EDS) simulation. The first crystal structure of catalytically deficient SpOPB (SpOPBS532A) with an intact hinge sequence is reported. Similarly to SpOPB with modified hinges, SpOPBS532A was crystallized in the presence of spermine and adopted an intermediate conformation in the crystal lattice. Despite the similarity of the crystal structures, a difference in the catalytic triad residue arrangement was detected, which explained the inhibitory effect of the hinge modification. The SpOPBS532A structure reconstituted to the wild-type form was used as a starting point to the classical MD followed by EDS simulation, which allowed us to simulate the domain separation and the transition of the enzyme from the intermediate to open conformation. The obtained open state model was in good agreement with the experimental SAXS data

Crystal Structure of Inhibitor-Bound Bacterial Oligopeptidase B in the Closed State: Similarity and Difference between Protozoan and Bacterial Enzymes

The crystal structure of bacterial oligopeptidase B from Serratia proteamaculans (SpOpB) in complex with a chloromethyl ketone inhibitor was determined at 2.2 Å resolution. SpOpB was crystallized in a closed (catalytically active) conformation. A single inhibitor molecule bound simultaneously to the catalytic residues S532 and H652 mimicked a tetrahedral intermediate of the catalytic reaction. A comparative analysis of the obtained structure and the structure of OpB from Trypanosoma brucei (TbOpB) in a closed conformation showed that in both enzymes, the stabilization of the D-loop (carrying the catalytic D) in a position favorable for the formation of a tetrahedral complex occurs due to interaction with the neighboring loop from the β-propeller. However, the modes of interdomain interactions were significantly different for bacterial and protozoan OpBs. Instead of a salt bridge (as in TbOpB), in SpOpB, a pair of polar residues following the catalytic D617 and a pair of neighboring arginine residues from the β-propeller domain formed complementary oppositely charged surfaces. Bioinformatics analysis and structural modeling show that all bacterial OpBs can be divided into two large groups according to these two modes of D-loop stabilization in closed conformations

Crystal Structure of Inhibitor-Bound Bacterial Oligopeptidase B in the Closed State: Similarity and Difference between Protozoan and Bacterial Enzymes

The crystal structure of bacterial oligopeptidase B from Serratia proteamaculans (SpOpB) in complex with a chloromethyl ketone inhibitor was determined at 2.2 Å resolution. SpOpB was crystallized in a closed (catalytically active) conformation. A single inhibitor molecule bound simultaneously to the catalytic residues S532 and H652 mimicked a tetrahedral intermediate of the catalytic reaction. A comparative analysis of the obtained structure and the structure of OpB from Trypanosoma brucei (TbOpB) in a closed conformation showed that in both enzymes, the stabilization of the D-loop (carrying the catalytic D) in a position favorable for the formation of a tetrahedral complex occurs due to interaction with the neighboring loop from the β-propeller. However, the modes of interdomain interactions were significantly different for bacterial and protozoan OpBs. Instead of a salt bridge (as in TbOpB), in SpOpB, a pair of polar residues following the catalytic D617 and a pair of neighboring arginine residues from the β-propeller domain formed complementary oppositely charged surfaces. Bioinformatics analysis and structural modeling show that all bacterial OpBs can be divided into two large groups according to these two modes of D-loop stabilization in closed conformations