Amino acid substitutions in human growth hormone affect secondary structure and receptor binding

PLOS ONE RESEARCH ARTICLE Amino acid substitutions in human growth hormone affect secondary structure and receptor binding Andrei Rajkovic1, Sandesh Kanchugal1, Eldar Abdurakhmanov2, Rebecca Howard3, Sebastian Wärmländer3, Joseph Erwin ID3, Hugo A. Barrera Saldaña4, Astrid Gräslund3, Helena Danielson ID2, Samuel Coulbourn Flores ID3,5* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden, 2 Department of Chemistry, Uppsala University, Uppsala, Sweden, 3 Department of Biochemistry and Biophysics, Stockholm University, Frescati, Sweden, 4 Department of Biochemistry, Autonomous University of Nuevo León, Monterrey, Mexico, 5 Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden * Abstract OPEN ACCESS Citation: Rajkovic A, Kanchugal S, Abdurakhmanov E, Howard R, Wärmländer S, Erwin J, et al. (2023) Amino acid substitutions in human growth hormone affect secondary structure and receptor binding. PLoS ONE 18(3): e0282741. https://doi. org/10.1371/journal.pone.0282741 Editor: Sabato D’Auria, Consiglio Nazionale delle Ricerche, ITALY Received: December 9, 2022 Accepted: February 22, 2023 Published: March 23, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0282741 Copyright: © 2023 Rajkovic et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. The interaction between human Growth Hormone (hGH) and hGH Receptor (hGHR) has basic relevance to cancer and growth disorders, and hGH is the scaffold for Pegvisomant, an anti-acromegaly therapeutic. For the latter reason, hGH has been extensively engineered by early workers to improve binding and other properties. We are particularly interested in E174 which belongs to the hGH zinc-binding triad; the substitution E174A is known to significantly increase binding, but to now no explanation has been offered. We generated this and several computationally-selected single-residue substitutions at the hGHR-binding site of hGH. We find that, while many successfully slow down dissociation of the hGHhGHR complex once bound, they also slow down the association of hGH to hGHR. The E174A substitution induces a change in the Circular Dichroism spectrum that suggests the appearance of coiled-coiling. Here we show that E174A increases affinity of hGH against hGHR because the off-rate is slowed down more than the on-rate. For E174Y (and certain mutations at other sites) the slowdown in on-rate was greater than that of the off-rate, leading to decreased affinity. The results point to a link between structure, zinc binding, and hGHR-binding affinity in hGH. Introduction Human Growth Hormone (hGH) binds a single hGH Receptor (hGHR) using its Site 1, a large, physicochemically diverse binding region. It then recruits a second hGHR to bind at its lower-affinity Site 2 [1]. The hGHR dimerization initiates signaling through the JAK/STAT pathway. Thus one strategy to disrupt signaling is to prevent dimerization. This in turn can be done by destroying binding at site 2, which is easily effected with mutations such as G120K [2]. It is also useful to simultaneously strengthen binding at site 1 [3], and both were done in PLOS ONE | https://doi.org/10.1371/journal.pone.0282741 March 23, 2023 1 / 13 PLOS ONE Funding: We gratefully acknowledge support from the Swedish Foundation for International Cooperation in Research and Higher Education (STINT, IG2012-5157) to SF. The Lars Hierta Memorial Foundation provided salary support for AR. SF receives partial salary support from the Swedish Research Council grant VR-M 201606301, the National Research School in Medical Bioinformatics. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Amino acid substitutions in human growth hormone affect secondary structure and receptor binding Pegvisomant development. As part of that process the interesting substitution E174 was discovered which increases binding but whose mechanism could not be explained by Cunningham & Wells [4]. In this work we measure the kinetics and secondary-structural effects of that mutation, along with that of a different substitution at the same position (E174Y), a negative control (L52F, which is positioned far from position 174, and outside the helices), and the Wild Type (WT, the neutral control). Pegvisomant is a recombinant hGH (rhGH) which was affinity matured at site 1 using phage display, reaching an affinity 400-fold higher than WT [3]. However, this generated substitutions at 15 amino acid positions in site 1, and other considerations required manually selected reversions and mutagenesis. The G120K mutation was also generated. Lastly, the rhGH was PEGylated to extend serum half-life. These manipulations resulted in significant reduction of affinity compared to WT [5]. As a result, there remains significant potential for rhGH variants to be used as therapeutics and diagnostics for several cancers and growth disorders, hence motivation to understand the biophysics of binding at site 1. We are interested in the possibility of choosing amino acid substitutions at a small number of residue positions, obtaining higher affinity while also working within constraints such as maintaining solubility and enabling modifications such as PEGylation. One could in principle also avoid patented substitutions, though that is not a concern in the case of Pegvisomant. Cunningham & Wells discovered E174A during alanine scanning—a technique used to map binding interfaces in the absence of structural data. Alanine is smaller than all other canonical amino acids except glycine. Therefore protein-protein interactions (PPIs) are usually weakened if the substitution is at the interface. However E174A is an exception–it increases affinity [4]. E174, along with H18 and H21, is part of a zinc-binding triad [6] (Fig 1). E174 is also packed between two helices and follows the typical heptad repeat [7]. We therefore paid particular attention to E174. How could an amino acid which is part of the zinc binding triad, be mutated (to alanine, no less) and increase affinity? More broadly, we are also interested in the general problem of computationally engineering proteins by introducing single-residue-position substitut (...truncated)