Testosterone meets albumin – the molecular mechanism of sex hormone transport by serum albumins†

[madman]

Serum albumin is the most abundant protein in mammalian blood plasma and is responsible for the transport of metals, drugs, and various metabolites, including hormones. We report the first albumin structure in complex with testosterone, the primary male sex hormone. Testosterone is bound in two sites, neither of which overlaps with the previously suggested Sudlow site I. We determined the binding constant of testosterone to equine and human albumins by two different methods: tryptophan fluorescence quenching and ultrafast affinity extraction. The binding studies and similarities between residues comprising the binding sites on serum albumins suggest that testosterone binds to the same sites on both proteins. Our comparative analysis of albumin complexes with hormones, drugs, and other biologically relevant compounds strongly suggests interference between a number of compounds present in blood and testosterone transport by serum albumin. We discuss a possible link between our findings and some phenomena observed in human patients, such as low testosterone levels in diabetic patients


The experimental determination of testosterone binding sites reported here sheds new light on the topic of drug delivery and possible interference between testosterone and various compounds transported in the bloodstream. First, both TBS1 and TBS2 have previously been shown to bind various hydrophilic and hydrophobic molecules. In TBS1, ESA has been shown to bind etodolac (PDB ID: 5V0V), (S)-cetirizine11 (PDB ID: 5DQF), a phosphorodithioate derivative of myristoyl cyclic phosphatidic acid (Myr-2S-cPA) (PDB ID: 5ID9), and naproxen (PDB ID: 4OT2). HSA has been shown to bind ibuprofen (PDB ID: 2BXG), diflunisal (PDB ID: 2BXE), and halothane (PDB ID: 1E7B) at this location as well. Molecules previously shown to bind to SA at TBS2 are (R)-cetirizine (PDB ID: 5DQF), Tris, and the acetate and malonate ions. The overlapping of testosterone binding sites with those of various drugs suggests possible interference between transport of drugs and that of testosterone. Multiple drugs have been shown to decrease testosterone levels. For example, a retrospective cohort study 58 and a meta-analysis 59 showed that male opioid users had significantly lower testosterone levels than control subjects. Additionally, statins (drugs used to lower cholesterol levels in the blood) have been shown to lower testosterone levels in middle-aged hypercholesterolemic men; however, the main expected reason for this effect is lower levels of cholesterol, which is a precursor to testosterone. The mechanisms that lead to decreased testosterone levels in these cases are not fully understood but could be partially explained by the interference between drug and testosterone transport.


Second, non-enzymatic glycosylation (glycation), which is promoted in diabetic patients, may affect HSA and alter its drug-binding ability. A number of lysine residues are glycated in HSA in vitro, among which Lys136 and Lys162 (Lys161 in ESA) are located in TBS2, and Lys351 (Lys350 in ESA) is located in TBS1. Arg209 (Arg208 in ESA) in TBS1 can also undergo glycation-related modifications. Therefore, it is possible that testosterone binding may be affected upon glycation of SA. (R)-Cetirizine, which binds in TBS211, has been shown to exhibit stronger binding to glycated HSA than to the non-glycated form. Because of SA's glycation, cetirizine administration in diabetic patients may need to be distinct from the standard dose in order to achieve the same curative effect. Due to the overlap of cetirizine and testosterone binding sites and the similarities of the ligand–residue interactions, we speculate that the relationship between low testosterone levels and insulin resistance in men with type 2 diabetes may be related to the glycation of residues comprising TBS1 and TBS2.


Third, the overlap of TBS1 with fatty acid binding site 6 (FA6) suggests that fatty acid levels in the blood might affect testosterone binding to SA. The presence of free fatty acids has been shown to inhibit testosterone binding to both SA and SHBG, increasing the fraction of free testosterone available for uptake by tissues. However, Watanabe et al. showed that the binding of free fatty acids to bovine serum albumin (BSA) increased the binding strength of testosterone. Thus, the effect of bound fatty acids is not completely known and may prove critical in furthering our understanding of the bioavailability of albumin bound testosterone, which remains controversial. In addition, testosterone is known to affect fatty acid concentrations via hormonal regulation; namely, the administration of testosterone has been shown to suppress regulatory enzymes in fatty acid synthesis, protect against hepatic steatosis (fatty liver), and decrease adiposity.


The first structure of SA in complex with a steroid sex hormone indicates that testosterone does not bind in the previously suggested first Sudlow site but rather in two sites known to bind fatty acids and drugs. The molecular details of the albumin–testosterone interaction suggest a potential competition between hormones, other metabolites, and drugs for binding to SA. These findings may impact future biomedical investigations of the SA–hormone interaction and have potential clinical implications for the development of new therapeutic agents.

 

[madman]

Fig. 1 Testosterone binding sites and their distances from the tryptophan residue. Distances were calculated between the center of mass of tryptophan's indole ring center of mass and that of each testosterone molecule. The testosterone molecules (yellow) and the tryptophan's side chain (blue) are shown in stick representation and labeled.

 

[madman]

Fig. 2 Testosterone binding sites with omit electron density map (mFo–DFc map, calculated after 10 refinement cycles without testosterone, σ – 3.0) presented in green and red (positive and negative contours, respectively). TBS1 is located between subdomains IIA and IIB, while TBS2 is surrounded by subdomains IA and IB. Colors of helices correspond with the domains' colors in Fig. 5. The electron density can be inspected interactively at https://molstack.bioreproducibility.org/p/9CP6/.

 

[madman]

Fig. 3 Hydrophobic nature of testosterone binding sites. The color of the protein surface indicates the contributions of the particular atoms to the surface. The color scheme is as follows: gray for carbon atoms, red for oxygen atoms, and blue for nitrogen atoms. Testosterone's carbon atoms are shown in yellow.

 

[madman]

Testosterone Discovery May Explain Low Levels in Men With Diabetes
March 27 2019




https://newsroom.uvahealth.com/2019...-may-explain-low-levels-in-men-with-diabetes/




Potential Answers About Low T
The discovery represents a collaboration of Minor’s lab, in UVA’s Department of Molecular Physiology and Biological Physics, with that of David Hage, PhD, of the Department of Chemistry at the University of Nebraska–Lincoln. Together, they developed the first 3D structure of serum albumin bound to a sex hormone with results of binding studies, which led to some unexpected findings.

Using an approach known as X-ray crystallography, the researchers were able to inspect the complex in unbelievable detail. But this was no easy task: First they had to capture the pairing inside a lab-made crystal – a tremendous challenge – and then they could bombard it with a stream of powerful X-rays. By tracking how the X-rays diffracted, they could determine the shape of something far too small for even the most powerful light microscopes to see.

What they found defied expectations: Testosterone was not bound in its previously predicted binding site but rather in two other sites, both with an affinity for fatty acids, various metabolites and certain drugs. That suggests that testosterone faces strong competition for transport inside the body – important information for doctors not just when prescribing existing treatments but also when looking for new treatments. (Serum albumin, for example, is an important carrier of cancer drugs.)