Rational cytokine design for increased lifetime and enhanced potency using pH-activated “histidine switching”

130

114

Erythropoietin (Epo) is essential for the production of mature red blood cells, and recombinant Epo is commonly used to treat anemia, but how Epo is degraded and cleared from the body is not understood. Glycosylation of Epo is required for its in vivo bioactivity, although not for in vitro receptor binding or stimulation of Epo-dependent cell lines; Epo glycosylation actually reduces the affinity of Epo for the Epo receptor (EpoR). Interestingly, a hyperglycosylated analog of Epo, called novel erythropoiesis-stimulating protein (NESP), has a lower affinity than Epo for the EpoR but has greater in vivo activity and a longer serum half-life than Epo. We hypothesize that a major mechanism for degradation of Epo in the body occurs in cells expressing the Epo receptor, through receptor-mediated endocytosis of Epo followed by degradation in lysosomes, and therefore investigated the trafficking and degradation Erythropoietin (Epo)2 and the erythropoietin receptor (EpoR) are required for the production of mature red blood cells (1); recombinant Epo is commonly used to treat anemia in cases of chronic renal failure, cancer, and AIDS. Epo contains one O-linked and three N-linked carbohydrate chains, each having 2-4 branches that often end in a negatively charged sialic acid. These carbohydrate chains are not required for receptor binding in vitro or stimulation of growth of EpoR-expressing cultured cells but are required for the in vivo bioactivity of Epo to increase red blood cell mass (2). Heterogeneous branching of Epo N-linked carbohydrates results in Epo isoforms with different sialic acid contents up to a maximum of 14. Epo isoforms with higher sialic acid content have a lower affinity for EpoR but a longer serum half-life and are more effective for stimulating the production of red blood cells in vivo (3).How Epo is cleared from the circulation and degraded in the body is not understood (4). In addition, we do not know how the sialic acid content of Epo has such a large effect on its serum half-life and in vivo bioactivity. Epo, both produced endogenously or injected, can be cleared from the circulation by filtration into urine. However, only a small fraction of an injected dose of Epo is excreted intact in urine (5); most of the Epo is degraded in the body, and the degradation products of Epo are excreted in urine (6). Where and how this degradation of Epo occurs is not known, and the clearance of Epo from the circulation and degradation in vivo may occur through more than one mechanism. It is possible that extracellular proteases and enzymes could degrade or modify Epo or that Epo could be taken up through unknown mechanisms and degraded in cells that do not express EpoR. However, there is a strong correlation between the affinity of Epo or Epo analogs for the EpoR and the in vivo lifetime of the ligand. In particular, a hyperglycosylated analog of Epo, called novel erythropoiesis-stimulating protein (NESP), was engineered to contain two additional N-linked carbohydrate chains and a maximum of 22 sialic acids (7...

Section: Discussionmentioning

confidence: 99%

Cellular Trafficking and Degradation of Erythropoietin and Novel Erythropoiesis Stimulating Protein (NESP)

Gross

Lodish

2006

130

114

“…PB calculations have been used successfully to engineer proteins with higher stability (34,35), but to best of our knowledge the same method as here has not been used much for engineering higher affinity for ligand binding. A related Tanford-Kirkwood electrostatic model (36) has been used in design of specificity of coiled-coil recognition, whereas a previous study (37) used continuum calculations to design cytokine variants with pH-dependent loss of affinity by a histidine-switch. The most significant results on affinity improvement via electrostatic optimization have been obtained by Schreiber and co-workers, who showed that the coloumbic interaction component of the electrostatic energy for encounter complex can be used for optimization of the on on-rate of binding (38).…”

Section: Discussionmentioning

confidence: 99%

Electrostatic Interactions of Hsp-organizing Protein Tetratricopeptide Domains with Hsp70 and Hsp90

Kajander

Sachs

Goldman

et al. 2009

The Hsp-organizing protein (HOP) binds to the C termini of the chaperones Hsp70 and Hsp90, thus bringing them together so that substrate proteins can be passed from Hsp70 to Hsp90. Because Hsp90 is essential for the correct folding and maturation of many oncogenic proteins, it has become a significant target for anti-cancer drug design. HOP binds to Hsp70 and Hsp90 via two independent tetratricopeptide (TPR) domains, TPR1 and TPR2A, respectively. We have analyzed ligand binding using Poisson-Boltzmann continuum electrostatic calculations, free energy perturbation, molecular dynamics simulations, and site-directed mutagenesis to delineate the contribution of different interactions to the affinity and specificity of the TPR-peptide interactions. We found that continuum electrostatic calculations could be used to guide protein design by removing unfavorable interactions to increase binding affinity, with an 80-fold increase in affinity for TPR2A. Contributions at buried charged residues, however, were better predicted by free energy perturbation calculations. We suggest using a combination of the two approaches for increasing the accuracy of results, with free energy perturbation calculations used only at selected buried residues of the ligand binding pocket. Finally we present the crystal structure of TPR2A in complex with its non-cognate Hsp70 ligand, which provides insight on the origins of specificity in TPR domain-peptide recognition.The Hsp-organizing protein (HOP) 4 plays a key role in in vivo folding by bringing the chaperones Hsp70 (or its constitutively expressed isoform, Hsp70) and Hsp90 together into a complex (1) (see Fig. 1), where specific substrate proteins are passed over from Hsp70 to Hsp90. Partially folded Hsp90 client proteins are transferred from Hsp70 to Hsp90, where the final steps of folding are completed (2, 3). While bound to the Hsp90, the client protein then becomes functional, e.g. hormone receptors can bind their ligands, and then are released. Both chaperones must be brought together by their interaction with HOP to accomplish this (see Fig. 1). The whole Hsp90 functional cycle involves also other co-chaperones, such as p23, other TPR proteins, and ATP binding and hydrolysis, and is fairly complicated and incompletely understood (4). However, it is clear that HOP facilitates the interaction of Hsp70 and Hsp90 and thus is important in providing the link between the chaperones in the early part of Hps90 functional cycle. Hsp90 client proteins include nuclear hormone receptors, p53, and other transcription factors and various kinases, such as Atk and Her2. Many of these are oncogenes, making Hsp90 a significant anti-cancer drug target (5). It is therefore of considerable importance to better understand the chaperone cycle of Hsp90 and in particular its interaction with essential co-chaperones, such as HOP. The interactions of Hsp70 and Hsp90 with HOP also provide an excellent model system in which to study the contribution of electrostatics to protein-peptide interactions, as will bec...

“…Histidine protonation can control the cytokine interactions between the cell surface and the endosomal pH (11). Protonation of histidine also induces the G protein conformational changes and drives membrane fusion (31).…”

Section: Discussionmentioning

confidence: 99%

Release Factors eRF1 and RF2

Nussinov

2004

Class I release factors 1 and 2 (RF1 and RF2) terminate protein synthesis by recognizing stop codons on the mRNA via their conserved amino acid motifs (NIKS in eRF1 and SPF in RF2) and by the conserved tripeptide (GGQ) interactions with the ribosomal peptidyltransferase center. Crystal structures of eRF1 and RF2 do not fit their ribosomal binding pocket (ϳ73 Å). Cryoelectron microscopy indicates large conformational changes in the ribosome-bound RF2. Here, we investigate the conformational dynamics of the eRF1 and RF2 using molecular dynamics simulation, structural alignment, and electrostatic analysis of domain interactions. We show that relaxed eRF1 has a shape remarkably similar to the ribosome-bound RF2 observed by cryoelectron microscopy. The similarity between the two release factors is as good as between elongation factor G and elongation factor Tu Protein synthesis on the ribosome has four steps: initiation, elongation, termination, and recycling. Class I release factors (RF1 and RF2) 1 terminate protein synthesis by recognizing stop codons on the mRNA (1) via their conserved amino acid motifs (NIKS in eRF1 and SPF in RF2) and by the conserved tripeptide (GGQ) interactions with the ribosomal peptidyltransferase center (2, 3). Prokaryotes have both RF1 and RF2, whereas eukaryotes have only one omnipotent factor, eRF1. Class II release factors RF3 and eRF3 have different functions, including the release of the Class I factors. Despite the fact that polypeptide release does not depend on tRNAs, it was suspected that Class I release factors (RF) mimic tRNAs (2, 4 -9), due to the functions of the two important motifs: stop codon recognition and peptide release activity. Several translation factors like the ribosomal recycling factor (RRF) and elongation factor G (EF-G) have shapes similar to that of tRNA (10 -12). However, unlike the tRNA conformity (13,14), where some structural features make the tRNAs equivalent and thus recognized by the translation apparatus (13,15,16), the available structures of the Class I release factors indicate that the RF conformity requires significant conformational changes.The distance between the ribosomal decoding center and the peptidyltransferase center is ϳ73 Å (2, 6, 7, 16). However, in the release factor crystal structures (4, 5), the separation between the NIKS and the GGQ motifs in eRF1 is 97.5 Å (measured from the Ile-C ␣ to Gln-C ␣ ), and the distance between the SPF and GGQ in RF2 is only 32 Å (Pro-C ␣ to Gln-C ␣ ). Two cryoelectron microscopy (cryo-EM) studies revealed large conformational changes upon binding of the RF2 to the ribosome (6, 7). The separation between SPF and GGQ in RF2 extended to 61 Å in one cryo-EM study (6) and to 73 Å in another cryo-EM study (7). The overall binding of the RF2 does not mimic tRNA, and it appears that the functional mimicry of the protein and the RNA is more convincing than a simple structural mimicry (2,8,9). Still, the distances between the two RNA signal binding motifs have to be around 73 Å for the Class I release factors. I...