A multi-step, dynamic allosteric model of testosterone's binding to sex hormone binding globulin.

PURPOSE: Circulating free testosterone (FT) levels have been used widely in the diagnosis and treatment of hypogonadism in men. Due to experimental complexities in FT measurements, the Endocrine Society has recommended the use of calculated FT (cFT) as an appropriate approach for estimating FT. We show here that the prevailing model of testosterone's binding to SHBG, which assumes that each SHBG dimer binds two testosterone molecules and that the two binding sites on SHBG have similar binding affinity is erroneous and provides FT values that differ substantially from those obtained using equilibrium dialysis. METHODS: We characterized testosterone's binding to SHBG using binding isotherms, ligand depletion curves, and isothermal titration calorimetry (ITC). We derived a new model of testosterone's binding to SHBG from these experimental data and used this model to determine FT concentrations and compare these values with those derived from equilibrium dialysis. RESULTS: Experimental data on testosterone's association with SHBG generated using binding isotherms including equilibrium binding, ligand depletion experiments, and ITC provide evidence of a multi-step dynamic process, encompassing at least two inter-converting microstates in unliganded SHBG, readjustment of equilibria between unliganded states upon binding of the first ligand molecule, and allosteric interaction between two binding sites of SHBG dimer. FT concentrations in men determined using the new multistep dynamic model with complex allostery did not differ from those measured using equilibrium dialysis. Systematic error in calculated FT vales in females using Vermeulen's model was also significantly reduced. In European Male Aging Study, the men deemed to have low FT (<2.5th percentile) by the new model were at increased risk of sexual symptoms and elevated LH. CONCLUSION: Testosterone's binding to SHBG is a multi-step dynamic process that involves complex allostery within SHBG dimer. FT values obtained using the new model have close correspondence with those measured using equilibrium dialysis.

Research resource: EPSLiM: ensemble predictor for short linear motifs in nuclear hormone receptors.

Nuclear receptors (NRs) are a superfamily of transcription factors central to regulating many biological processes, including cell growth, death, metabolism, and immune responses. NR-mediated gene expression can be modulated by coactivators and corepressors through direct physical interaction or protein complexes with functional domains in NRs. One class of these domains includes short linear motifs (SLiMs), which facilitate protein-protein interactions, phosphorylation, and ligand binding primarily in the intrinsically disordered regions (IDRs) of proteins. Across all proteins, the number of known SLiMs is limited due to the difficulty in studying IDRs experimentally. Computational tools provide a systematic and data-driven approach for predicting functional motifs that can be used to prioritize experimental efforts. Accordingly, several tools have been developed based on sequence conservation or biophysical features; however, discrepancies in predictions make it difficult to determine the true candidate SLiMs. In this work, we present the ensemble predictor for short linear motifs (EPSLiM), a novel strategy to prioritize the residues that are most likely to be SLiMs in IDRs. EPSLiM applies a generalized linear model to integrate predictions from individual methodologies. We show that EPSLiM outperforms individual predictors, and we apply our method to NRs. The androgen receptor is an example with an N-terminal domain of 559 disordered amino acids that contains several validated SLiMs important for transcriptional activation. We use the androgen receptor to illustrate the predictive performance of EPSLiM and make the results of all human and mouse NRs publically available through the web service http://epslim.bwh.harvard.edu.

Multifunctional magnetic rotator for micro and nanorheological studies.

We report on the development of a multifunctional magnetic rotator that has been built and used during the last five years by two groups from Clemson and Drexel Universities studying the rheological properties of microdroplets. This magnetic rotator allows one to generate rotating magnetic fields in a broad frequency band, from hertz to tens kilohertz. We illustrate its flexibility and robustness by conducting the rheological studies of simple and polymeric fluids at the nano and microscale. First we reproduce a temperature-dependent viscosity of a synthetic oil used as a viscosity standard. Magnetic rotational spectroscopy with suspended nickel nanorods was used in these studies. As a second example, we converted the magnetic rotator into a pump with precise controlled flow modulation. Using multiwalled carbon nanotubes, we were able to estimate the shear modulus of sickle hemoglobin polymer. We believe that this multifunctional magnetic system will be useful not only for micro and nanorheological studies, but it will find much broader applications requiring remote controlled manipulation of micro and nanoobjects.

Dynamics of coregulator-induced conformational perturbations in androgen receptor ligand binding domain.

Androgen receptor (AR) coregulators modulate ligand-induced gene expression in a tissue specific manner. The molecular events that follow coactivator binding to AR and the mechanisms that govern the sequence-specific effects of AR coregulators are poorly understood. Using consensus coactivator sequence D11-FxxLF and biophysical techniques, we show that coactivator association is followed by conformational rearrangement in AR ligand binding domain (AR-LBD) that is enthalpically and entropically favorable with activation energy of 29.8±4.2 kJ/mol. Further characterization of ARA70 and SRC3-1 based consensus sequences reveal that each coactivator induces a distinct conformational state in the dihydrotestosterone:AR-LBD:coactivator complex. Complementary computational modeling revealed that coactivator induced specific alterations in the backbone flexibility of AR-LBD distant from the site of coactivator binding and that the intramolecular rearrangements in AR-LBD backbone induced by the two coactivator peptides were different. These data suggest that coactivators may impart specificity in the transcriptional machinery by changing the steady-state conformation of AR-LBD. These data provide direct evidence that even in the presence of same ligand, AR-LBD can occupy distinct conformational states depending on its interactions with specific coactivators in the tissues. We posit that this coactivator-specific conformational gating may then dictate subsequent binding partners and interaction/affinity for the DNA-response elements.

Pressure perturbation calorimetry of lipoproteins reveals an endothermic transition without detectable volume changes. Implications for adsorption of apolipoprotein to a phospholipid surface.

Plasma lipoproteins are assemblies of lipids and apolipoproteins that mediate lipid transport and metabolism. High-density lipoproteins (HDL) remove excess cell cholesterol and provide protection against atherosclerosis. Important aspects of metabolic HDL remodeling, including apolipoprotein dissociation and lipoprotein fusion, are mimicked in thermal denaturation. We report the first study of the protein-lipid complexes by pressure perturbation calorimetry (PPC) beyond 100 °C. In PPC, volume expansion coefficient α(v)(T) is measured during heating; in proteins, α(v)(T) is dominated by hydration. Calorimetric studies of reconstituted HDL and of human high-density, low-density, and very low-density lipoproteins reveal that apolipoprotein unfolding, dissociation, and lipoprotein fusion are endothermic transitions without detectable volume changes. This may result from the limited applicability of PPC to slow kinetically controlled transitions such as thermal remodeling of lipoproteins and/or from the possibility that this remodeling causes no significant changes in the solvent structure and, hence, may not involve large transient solvent exposure of apolar moieties. Another conclusion is that apolipoprotein A-I in solution adsorbs to the phospholipid surface; protein hydration is preserved upon such adsorption. We posit that adsorption to a phospholipid surface helps recruit free apolipoprotein to the plasma membrane and facilitate HDL biogenesis.

The dynamic structure of the estrogen receptor.

The estrogen receptor (ER) mediates most of the biological effects of estrogens at the level of gene regulation by interacting through its site-specific DNA and with other coregulatory proteins. In recent years, new information regarding the dynamic structural nature of ER has emerged. The physiological effects of estrogen are manifested through ER's two isoforms, ER(α) and ER(ß). These two isoforms (ER(α) and ER(ß)) display distinct regions of sequence homology. The three-dimensional structures of the DNA-binding domain (DBD) and ligand-binding domain (LBD) have been solved, whereas no three-dimensional natively folded structure for the ER N-terminal domain (NTD) is available to date. However, insights about the structural and functional correlations regarding the ER NTD have recently emerged. In this paper, we discuss the knowledge about the structural characteristics of the ER in general and how the structural features of the two isoforms differ, and its subsequent role in gene regulation.

The microrheology of sickle hemoglobin gels.

Sickle cell disease is a rheological disease, yet no quantitative rheological data exist on microscopic samples at physiological concentrations. We have developed a novel method for measuring the microrheology of sickle hemoglobin gels, based on magnetically driven compression of 5- to 8-microm-thick emulsions containing hemoglobin droplets approximately 80 microm in diameter. Using our method, by observing the expansion of the droplet area as the emulsion is compressed, we were able to resolve changes in thickness of a few nanometers with temporal resolution of milliseconds. Gels were formed at various initial concentrations and temperatures and with different internal domain structure. All behaved as Hookean springs with Young's modulus from 300 to 1500 kPa for gels with polymerized hemoglobin concentration from 6 g/dl to 12 g/dl. For uniform, multidomain gels, Young's modulus mainly depended on the terminal concentration of the gel rather than the conditions of formation. A simple model reproduced the quadratic dependence of the Young's modulus on the concentration of polymerized hemoglobin. Partially desaturated samples also displayed quadratic concentration dependence but with a smaller proportionality coefficient, as did samples that were desaturated in steps; such samples were significantly less rigid than gels formed all at once. The magnitude of the Young's modulus provides quantitative support for the dominant models of sickle pathophysiology.