Clathrin-mediated endocytosis (CME) is crucial for regulating G protein-coupled receptors (GPCRs) via phosphorylation-dependent arrestin interactions. Despite detailed structural knowledge on the arrestin interactions with phosphorylated tails of GPCRs, the interplay between receptor phosphorylation and arrestin coupling to the CME machinery is not well understood, in particular due to the weakness and dynamics of the individual molecular interactions. Here, we have characterized the interactions of arrestin2, which is activated by the phosphorylated C-terminus of the human chemokine receptor 5 (CCR5), with the main protein constituents of CME, namely clathrin and AP2, by solution NMR spectroscopy, biochemical, and cellular assays. The NMR analysis revealed that arrestin2 interacts weakly with clathrin through a single binding site, independent of arrestin2 activation. In contrast, the arrestin2-AP2 interaction is stronger, requires arrestin2 activation by the CCR5 phospho-tail, and depends quantitatively on its degree of phosphorylation. These in vitro results are corroborated by cellular assays, which show that the chemokine-induced formation of a long-lived CCR5-arrestin2 internalization complex depends strongly on the interaction of arrestin2 with AP2, but not with clathrin. Taken together, these findings provide quantitative, atom-scale insights on the first steps of CCR5 endocytosis.

Arrestins are essential proteins for the regulation of G protein-coupled receptors (GPCRs). They mediate GPCR desensitization after the activated receptor has been phosphorylated by G protein receptor kinases (GRKs). In addition, GPCR-arrestin interactions may trigger signaling pathways that are distinct and independent from G proteins. The non-visual GPCRs encompass hundreds of receptors with varying phosphorylation patterns and amino acid sequences, which are regulated by only two human non-visual arrestin isoforms. This review describes recent findings on GPCR-arrestin complexes, obtained by structural techniques, biophysical, biochemical, and cellular assays. The solved structures of complete GPCR-arrestin complexes are of limited resolution ranging from 3.2 to 4.7 Å and reveal a high variability in the relative receptor-arrestin orientation. In contrast, biophysical and functional data indicate that arrestin recruitment, activation and GPCR-arrestin complex stability depend on the receptor phosphosite sequence patterns and density. At present, there is still a manifest lack of high-resolution structural and dynamical information on the interactions of native GPCRs with both GRKs and arrestins, which could provide a detailed molecular understanding of the genesis of receptor phosphorylation patterns and the specificity GPCR-arrestin interactions. Such insights seem crucial for progress in the rational design of advanced, arrestin-specific therapeutics.

The two non-visual arrestins, arrestin2 and arrestin3, bind hundreds of GPCRs with different phosphorylation patterns, leading to distinct functional outcomes. Structural information on these interactions is available only for very few GPCRs. Here, we have characterized the interactions between the phosphorylated human CC chemokine receptor 5 (CCR5) and arrestin2. We identified several new CCR5 phosphorylation sites necessary for stable arrestin2 complex formation. Structures of arrestin2 in the apo form and complexes with CCR5 C-terminal phosphopeptides, together with NMR, biochemical, and functional assays, revealed three phosphoresidues in a pXpp motif that are essential for arrestin2 binding and activation. The identified motif appears responsible for robust arrestin2 recruitment in many other GPCRs. An analysis of receptor sequences and available structural and functional information provides hints on the molecular basis of arrestin2/arrestin3 isoform specificity. Our findings demonstrate how multi-site phosphorylation controls GPCR⋅arrestin interactions and provide a framework to probe the intricate details of arrestin signaling.

The numerous chemokines and their cognate G protein-coupled chemokine receptors on the surface of leukocytes form a complex signaling network, which regulates the immune response and also other key physiological processes. Currently only a very limited number of structures of chemokine•chemokine receptor complexes have been solved. More structures are needed for the understanding of their mechanism of action and the rational design of drugs against these highly relevant therapeutic targets. Recently, we have determined the cryo-EM structure of the human wild-type CCR5 chemokine receptor, which is also the HIV-1 coreceptor, in its active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric Gi protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist CCR5 chemokine ligands. In this chapter, we present a detailed protocol for the preparation of the active agonist chemokine•CCR5•Gi complex for cryo-EM studies including quality controls and caveats. As such the protocol may serve as starting point for structural and biophysical studies of other chemokine•chemokine receptor complexes.

The human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in inflammation and is involved in cancer, HIV, and COVID-19. Despite its importance as a drug target, the molecular activation mechanism of CCR5, i.e., how chemokine agonists transduce the activation signal through the receptor, is yet unknown. Here, we report the cryo-EM structure of wild-type CCR5 in an active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric Gi protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist chemokines. The N terminus of agonist chemokines pushes onto specific structural motifs at the bottom of the orthosteric pocket that activate the canonical GPCR microswitch network. This activation mechanism differs substantially from other CC chemokine receptors that bind chemokines with shorter N termini in a shallow binding mode involving unique sequence signatures and a specialized activation mechanism.

It was found that oligo(hexamethylene guanidine) (OHMG) reacted very slowly with unactivated high-molecular-mass alkyl halides (dodecyl chloride), possibly due to steric hindrance and the low nucleophilicity of the guanidine moiety. Therefore, this alkyl halide could not be used to modify OHMG under conventional conditions. Ethylation of OHMG was much more facile. However, the substitution in the derivatives was broadly scattered (under ordinary conditions and reagent ratios). It was shown simultaneously that the antibacterial activity of the alkylated derivatives was less than that of the starting OHMG hydrochloride. Benzyl chloride reacted readily with OHMG to produce derivatives with the desired degree of substitution. It was found that benzyl derivatives with 0.2 degree of substitution were more active against mycobacteria than the starting hydrochloride with the activity gradually diminishing if the degree of substitution was increased further. The increased activity may have been related to the optimum increase of OHMG hydrophobicity and/or steric structure. The rather high antibacterial activities of the benzyl derivatives against Mycobacterium smegmatis strain ATCC 607 made them promising for further development and studies of the properties of alkylated OHMG derivatives to discover more active derivatives, including against M. smegmatis. The most obvious candidates could become the methylcarboxy-substituted OHMG derivatives.

Is necessary to ensure thorough purification of the target proteins, since the presence of host cell proteins (HCP) from CHO cells in the final product is fraught with a number of negative consequences. Thus, it is has to reduce the contamination of HCP to minimum levels. A comparison of the immunoenzymatic assays of the second and third generations of Cygnus technologies for the quantification of host cell proteins (CHO) in a monoclonal antibody adalimumab was done. It was found that a kit of the 2nd generation does not show the true level of the host cell proteins, underestimating the result. In this connection, it becomes necessary to confirm the validity of the method for determining the total quantity of host cell proteins (CHO) by a set of 3rd generation in adalimumab substance according to the following analytical characteristics: system suitability test, specificity, limit of detection, limit of quantitation, hook capacity, precision.

Рассмотрены подходы к созданию комплексов на основе разветвленного гидрохлорида олигогексаметиленгуанидина. Получены новые комбинации бактерицидных препаратов, подобраны методы синтеза ряда производных пара-аминосалициловой кислоты, исследована растворимость их комплексов с гидрохлоридом олигогексаметиленгуанидина, а также изучена бактерицидная активность полученных комплексов в отношении Mycobacterium smegmatis. Установлено, что минимальная подавляющая концентрация всех комплексов находится в пределах от 0,1 до 1 мкг/мл культуральной жидкости. Таким образом, полученные комплексы могут быть использованы для создания бактерицидного препарата с мощным биоцидным, пролонгированным действием, высокой воспроизводимостью свойств и низкой токсичностью.

Установлено, что олигогексаметиленгуанидин очень медленно реагирует с неактивированными алкилгалогенидами с высокой молекулярной массой (додецилхлорид), что, возможно, обусловлено стерическими затруднениями и низкой нуклеофильностью гуанидинового фрагмента, поэтому данный алкилгалогенид не может быть использован для модификации олигомера в обычных условиях. Этилированные производные олигомера получаются гораздо легче, однако они имеют сильный разброс степени замещения (при одинаковых условиях и соотношении реагентов). Одновременно показано, что при алкилировании антибактериальная активность производных понижается по сравнению с гидрохлоридом исходного олигомера. Бензилхлорид легко реагирует с олигомером и при этом получаются производные с желаемой степенью замещения. Установлено, что бензильные производные со степенью замещения 0,2 обладают повышенной активностью в отношении микобактерий по сравнению с исходным гидрохлоридом, при дальнейшем увеличении степени замещения активность постепенно снижается. Возможно, повышение активности связано с оптимальным увеличением гидрофобности и/или пространственной структуры олигомера. Наличие достаточно высокой антибактериальной активности бензильных производных в отношении Mycobacterium smegmatis штамм АТСС 607 делает перспективными дальнейшие работы по получению и изучению свойств алкилированых производных олигогесаметиленгуанидина с целью получения еще более активных производных, в т.ч. против M. smegmatis . Наиболее очевидными кандидатами могут стать метилкарбоксизамещенные производные олигомеров.

Approaches to creating complexes based on branched oligohexamethyleneguanidine hydrochloride are addressed. Novel combinations of bactericidal agents were obtained, methods for the synthesis of a series of p-aminosalicylic acid derivatives were selected, the solubilities of their complexes with oligohexamethyleneguanidine were determined, and the bactericidal activities of the resulting complexes against Mycobacterium smegmatis were assessed. The minimum inhibitory concentrations of all complexes were in the range 0.1 – 1 μg/ml of culture medium. Thus, these complexes may be useful for creating a bactericidal formulation with powerful and long-lasting biocidal action, highly reproducible properties, and low toxicity.