Biomedical subjects

James R Horn

Publications and source records attributed to James R Horn.

5 recordsLinked to original sources

PubMed2006

The role of protein dynamics in increasing binding affinity for an engineered protein-protein interaction established by H/D exchange mass spectrometry.

It is generally accepted that protein and solvation dynamics play fundamental roles in the mechanisms of protein-protein binding; however, assessing their contribution meaningfully has not been straightforward. Here, hydrogen/deuterium exchange mass spectrometry (H/D-Ex) was employed to assess the role of dynamics for a high-affinity human growth hormone variant (hGHv) and the wild-type growth hormone (wt-hGH) each binding to the extracellular domain of their receptor (hGHbp). Comparative analysis of the transient fluctuations in the bound and unbound states revealed that helix-1 of hGHv undergoes significant transient unfolding in its unbound state, a characteristic that was not found in wt-hGH or apparent in the temperature factor data from the X-ray analysis of the unbound hGHv structure. In addition, upon hormone binding, an overall increase in stability was observed for the beta-sheet structure of hGHbp which included sites distant from the binding interface. On the basis of the stability, binding kinetics, and thermodynamic data presented, the increase in the binding free energy of hGHv is primarily generated by factors that appear to increase the energy of the unbound state relative to the free energy of the bound complex. This implies that an alternate route to engineer new interactions aiming to increase protein-protein association energies may be achieved by introducing certain mutations that destabilize one of the interacting molecules without destabilizing the resulting bound complex. Importantly, although the hGHv molecule is less stable than its wt-hGH counterpart, its resulting active ternary complex with two copies of hGHbp has comparable stability to the wt complex.

Binding Sites↗

PubMed2005

Shotgun alanine scanning shows that growth hormone can bind productively to its receptor through a drastically minimized interface.

The high affinity binding site (Site1) of the human growth hormone (hGH) binds to its cognate receptor (hGHR) via a concave surface patch containing about 35 residues. Using 167 sequences from a shotgun alanine scanning analysis of Site1, we have determined that over half of these residues can be simultaneously changed to an alanine or a non-isosteric amino acid while still retaining a high affinity interaction. Among these hGH variants the distribution of the mutation is highly variable throughout the interface, although helix 4 is more conserved than the other binding elements. Kinetic and thermodynamic analyses were performed on 11 representative hGH Site1 variants that contained 14-20 mutations. Generally, the tightest binding variants showed similar associated rate constants (k(on)) as the wild-type (wt) hormone, indicating that their binding proceeds through a similar transition state intermediate. However, calorimetric analyses indicate very different thermodynamic partitioning: wt-hGH binding exhibits favorable enthalpy and entropy contributions, whereas the variants display highly favorable enthalpy and highly unfavorable entropy contributions. The heat capacities (DeltaCp) on binding measured for wt-hGH and its variants are significantly larger than normally seen for typical protein-protein interactions, suggesting large conformational or solvation effects. The multiple Site1 mutations are shown to indirectly affect binding of the second receptor at Site2 through an allosteric mechanism. We show that the stability of the ternary hormone-receptor complex reflects the affinity of the Site2 binding and is surprisingly exempt from changes in Site1 affinity, directly demonstrating that dissociation of the active signaling complex is a stepwise process.

Alanine↗

PubMed2004

Allosteric inhibition through core disruption.

Although inhibitors typically bind pre-formed sites on proteins, it is theoretically possible to inhibit by disrupting the folded structure of a protein or, in the limit, to bind preferentially to the unfolded state. Equilibria defining how such molecules act are well understood, but structural models for such binding are unknown. Two novel inhibitors of beta-lactamase were found to destabilize the enzyme at high temperatures, but at lower temperatures showed no preference for destabilized mutant enzymes versus stabilized mutants. X-ray crystal structures showed that both inhibitors bound to a cryptic site in beta-lactamase, which the inhibitors themselves created by forcing apart helixes 11 and 12. This opened up a portion of the hydrophobic core of the protein, into which these two inhibitors bind. Although this binding site is 16 A from the center of the active site, the conformational changes were transmitted through a sequence of linked motions to a key catalytic residue, Arg244, which in the complex adopts conformations very different from those in catalytically competent enzyme conformations. These structures offer a detailed view of what has heretofore been a theoretical construct, and suggest the possibility for further design against this novel site.

Allosteric Regulation↗

PubMed2003

Structure and energetics of protein-protein interactions: the role of conformational heterogeneity in OMTKY3 binding to serine proteases.

Proteins with flexible binding surfaces can interact with numerous binding partners. However, this promiscuity is more difficult to understand in "rigid-body" proteins, whose binding results in little, or no, change in the position of backbone atoms. The binding of Kazal inhibitors to serine proteases is considered a classic case of rigid-body binding, although they bind to a wide range of proteases. We have studied the thermodynamics of binding of the Kazal serine protease inhibitor, turkey ovomucoid third domain (OMTKY3), to the serine protease subtilisin Carlsberg using isothermal titration calorimetry and have determined the crystal structure of the complex at very high resolution (1.1A). Comparison of the binding energetics and structure to other OMTKY3 interactions demonstrates that small changes in the position of side-chains can make significant contributions to the binding thermodynamics, including the enthalpy of binding. These effects emphasize that small, "rigid-body" proteins are still dynamic structures, and these dynamics make contributions to both the enthalpy and entropy of binding interactions.

Amino Acid Sequence↗

PubMed2002

van't Hoff and calorimetric enthalpies II: effects of linked equilibria.

The complexity of binding reactions, including the linkage with other equilibria, is becoming increasingly apparent in biological processes such as signal transduction. Understanding these interactions requires obtaining thermodynamic profiles for each of the equilibria that occur in a binding event. Concern has been raised as to whether linked equilibria contribute differently to thermodynamics, such as DeltaH degrees and DeltaC(p), obtained from calorimetric and van't Hoff methods. We have previously shown that linked equilibria do not contribute differently to the van't Hoff and calorimetrically determined DeltaH degrees for processes such as linked folding or hydration. Here, examples of proton and ion linkage are examined. We show that there is no reason to expect the calorimetric and van't Hoff DeltaH degrees to be different, even without prior knowledge of the presence or absence of linked equilibria, as long as the system is permitted to equilibrate. However, it is possible to create experimental scenarios that result in and discrepancies. Furthermore, it is found that the presence of linked equilibria in all cases can result in "nonconventional" DeltaH degrees and DeltaC(p) profiles, making data analysis nontrivial.

Binding Sites↗