Crystal Structure and Site 1 Binding Energetics of Human Placental Lactogen

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In primates, placental lactogen (PL) is a pituitary hormone with fundamental roles during pregnancy involving fetal growth, metabolism, and stimulating lactation in the mother. Human placental lactogen (hPL) is highly conserved with human growth hormone (hGH) and both hormones bind to the hPRLR extracellular domain (ECD), the first step in receptor homodimerization, in a Zn2+-dependent manner. A modified surface plasmon resonance method was developed to measure the kinetics for hPL and hGH binding to the hPRLR ECD, with and without Zn2+ and showed that hPL has about a tenfold higher affinity for the hPRLR ECD1 than hGH. The crystal structure of the free state of hPL has been determined to 2.0 Å resolution showing the molecule possesses an overall structure similar to other long chain four-helix bundle cytokines. Comparison of the free hPL structure with the 1:1 complex structure of hGH bound to the hPRLR ECD1 suggests that two surface loops undergo conformational changes >10 Å upon binding. An 18 residue Ala-scan was used to characterize the binding energy epitope for the site 1 interface of hPL. Individual alanine substitutions at five positions reduced binding affinity by a ΔΔG≥3 kcal mol−1. A comparison of the hPL site 1 epitope with that previously determined for hGH indicates contributions of individual residues track reasonably well between hPL and hGH. In particular, residues involved in the zinc-binding site and Lys172 constitute the principal binding determinants for both hormones. However, several residues that are identical between hPL and hGH contribute quite differently to the binding of the hPRLR ECD1. Additionally, the overall magnitudes of the ΔΔG changes observed from the Ala-scan of hPL were markedly larger than those determined in the comparative scan of hGH to the hPRLR ECD1. The structural and biophysical data presented here show that subtle changes in the structural context of an interaction can lead to significantly different effects at the individual residue level.

Introduction

Placental lactogens (PL) and prolactins (PRL) are pituitary hormones with essential roles in a broad range of biological functions.1 Human placental lactogen (hPL), also called human chorionic somatomammotropin, is produced only during pregnancy and is involved in stimulating lactation, fetal growth and metabolism. During pregnancy in humans, both hPRL and growth hormone (hGH) are down-regulated. They are replaced by two other hormones: hPL, to induce prolactin-like activities, and a placental growth hormone, an hGH sequence variant that stimulates the GH receptor, but does not cross-react with the PRL receptor.2

The biological effects of PRL and GH are triggered via a hormone-induced receptor homodimerization process.3, 4 hGH activates both hGHR and hPRLR; however, although it shares 85% protein sequence identity with hGH, hPL does not interact with the hGHR, but activates solely the hPRLR, similar to hPRL (<25% protein sequence identity with hPL and hGH, Figure 1(a)). In order to bind to the hPRLR, both hPL and hGH use a Zn2+-binding site with the hPRLR; whereas, PRL does not.5, 6

Primate PRL receptor biology functions through extensive cross-reactivity, where biological effects can be induced by PRLs, PLs, and GHs.1 PLs produced by primates are derived from a GH lineage, while PLs from lower species are derived from a PRL origin.1, 7 The reason for this remains unclear. However, a protein engineering study demonstrated that substitution of the hGH sequence at five positions produced an hPL variant with a site 1 binding affinity similar to that of hGH to the hGHR extracellular domain 1 (ECD1).6 Thus, hPL represent a unique bridge to study the different molecular recognition properties between the PL and GH receptors signaling pathways.

There is a wide range of literature on cell biological, biophysical, structural, and protein engineering studies for the GH and PRL families;8, 9, 10 however, there have been fewer experimental studies of PLs from human and other species.6, 11, 12, 13 In particular, there is no information as to whether the PLs, which are expressed to perform their functions only during very specific situations (i.e. pregnancy), have unique binding characteristics compared to their close biological homologues, PRL and GH.

In order to further our knowledge of hPL biology from a structural and biophysical point of view, we have determined the crystal structure of the free state of hPL and compared it to hGH from the structure of the 1:1 complex of hGH:hPRLR ECD1.14 This comparison suggests that two regions of hPL undergo large conformational changes upon binding the hPRLR ECD1. It was known that both hPL and hGH bind the hPRLR ECD in a Zn2+-dependent fashion,5, 6 but the kinetics of the process have not been well characterized. To establish the effects of Zn2+ on the binding kinetics of hPL, hPRL, and hGH to the hPRLR ECD1, we have developed a modified surface plasmon resonance (SPR) method that overcomes the inherent non-specific binding effects that were induced by Zn2+. This allowed us to confirm the importance of Zn2+ for hPL and hGH binding, and the absence of a direct Zn2+ effect in the case of hPRL.

Using an 18 residue Ala-scan, we mapped the site 1 binding energy surface of hPL to the hPRLR ECD1. We found that identical residues in hPL and hGH exhibit different energetic contributions to binding the hPRLR ECD1. The overall magnitudes of the energy differences observed in the Ala-scan of hPL are generally systematically larger than those determined for the same residues in the comparative hGH Ala-scan. There are three residues in hPL that, when mutated to alanine, completely abolish binding; whereas, the same mutations in hGH show significant decreases in binding, but do not eliminate it. Interestingly, the observed effects of the single-site alanine mutations in both hPL and hGH relative to their binding to the hPRLR are generally significantly larger than those seen in the case of wild-type (wt)-hGH to its cognate receptor, hGHR.

Section snippets

hPL structure

The crystal structure of hPL was determined experimentally using multiwavelength anomalous dispersion (MAD) phasing methods.15 hPL is a 191 amino acid residue protein containing six methionine residues that were converted to seleomethionine to facilitate MAD phasing. The initial maps were calculated using experimental MAD phases to 2.7 Å. The data were extended incrementally and the updated model refined using a native high-resolution data set to 2.0 Å (Table 1). The final model was refined to a

Comparison of the site 1 binding hot-spot regions in hPL and hGH

As noted above, virtually all the changes in affinities for the hPL variants binding to either the hGHR or the hPRLR ECDs are due to differences in off-rates. This property suggests that the binding transition state energy is not affected significantly by factors at the level of the structural changes introduced by the mutations, even among variants that result in large decreases in binding affinity. A similar trend in binding kinetics has been seen for site 1 and site 2 binding variants of hGH

Sample preparation

Human placental lactogen and variants were expressed and purified as described.6 The ECD (residues 1–211) of the hPRLR receptor was expressed at 20 °C and purified as described.5 The free unpaired cysteine residue (C184) was mutated to alanine and serves as the wild-type receptor protein. The C184A-hPRLR does not affect binding affinity to the hormones.5 The ECD of the hGHR (residues 29–238) containing a S237C mutation was grown and purified as described.22, 36 All mutations were made using

Acknowledgements

We thank Drs B. Gopal, Julie Dohm, and Apostolos Gittis for advice and help with the crystallography. We thank the staffs of BioCARS and the Structural Biology Center for help with data collection. Use of the Advanced Photon Source was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under contract no. W-31-109-Eng-38. Use of the BioCARS Sector 14 was supported by the US National Institutes of Health (NIH), National Center for Research Resources, under grant

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