Insight into the allosteric mechanism of Scapharca dimeric hemoglobin.

Allosteric regulation is an essential function of many proteins that control a variety of different processes such as catalysis, signal transduction, and gene regulation. Structural rearrangements have historically been considered the main means of communication between different parts of a protein. Recent studies have highlighted the importance, however, of changes in protein flexibility as an effective way to mediate allosteric communication across a protein. Scapharca dimeric hemoglobin (HbI) is the simplest possible allosteric system, with cooperative ligand binding between two identical subunits. Thermodynamic equilibrium studies of the binding of oxygen to HbI have shown that cooperativity is an entropically driven effect. The change in entropy of the system observed upon ligand binding may arise from changes in the protein, the ligand, or the water of the system. The goal of this study is to determine the contribution of the change in entropy of the protein backbone to HbI cooperative binding. Molecular dynamics simulations and nuclear magnetic resonance relaxation techniques have revealed that the fast internal motions of HbI contribute to the cooperative binding to carbon monoxide in two ways: (1) by contributing favorably to the free energy of the system and (2) by participating in the cooperative mechanism at the HbI subunit interface. The internal dynamics of the weakly cooperative HbI mutant, F97Y, were also investigated with the same methods. The changes in backbone NH dynamics observed for F97Y HbI upon ligand binding are not as large as for the wild type, in agreement with the reduced cooperativity observed for this mutant. The results of this study indicate that interface flexibility and backbone conformational entropy of HbI participate in and are important for the cooperative mechanism of carbon monoxide binding.

Molecular mechanisms of viral and host cell substrate recognition by hepatitis C virus NS3/4A protease.

Hepatitis C NS3/4A protease is a prime therapeutic target that is responsible for cleaving the viral polyprotein at junctions 3-4A, 4A4B, 4B5A, and 5A5B and two host cell adaptor proteins of the innate immune response, TRIF and MAVS. In this study, NS3/4A crystal structures of both host cell cleavage sites were determined and compared to the crystal structures of viral substrates. Two distinct protease conformations were observed and correlated with substrate specificity: (i) 3-4A, 4A4B, 5A5B, and MAVS, which are processed more efficiently by the protease, form extensive electrostatic networks when in complex with the protease, and (ii) TRIF and 4B5A, which contain polyproline motifs in their full-length sequences, do not form electrostatic networks in their crystal complexes. These findings provide mechanistic insights into NS3/4A substrate recognition, which may assist in a more rational approach to inhibitor design in the face of the rapid acquisition of resistance.

Clusters of branched aliphatic side chains serve as cores of stability in the native state of the HisF TIM barrel protein.

Imidazole-3-glycerol phosphate synthase is a heterodimeric allosteric enzyme that catalyzes consecutive reactions in imidazole biosynthesis through its HisF and HisH subunits. The unusually slow unfolding reaction of the isolated HisF TIM barrel domain from the thermophilic bacteria, Thermotoga maritima, enabled an NMR-based site-specific analysis of the main-chain hydrogen bonds that stabilize its native conformation. Very strong protection against exchange with solvent deuterium in the native state was found in a subset of buried positions in α-helices and pervasively in the underlying ß-strands associated with a pair of large clusters of isoleucine, leucine and valine (ILV) side chains located in the α7(ßα)8(ßα)1-2 and α2(ßα)3-6ß7 segments of the (ßα)8 barrel. The most densely packed region of the large cluster, α3(ßα)4-6ß7, correlates closely with the core of stability previously observed in computational, protein engineering and NMR dynamics studies, demonstrating a key role for this cluster in determining the thermodynamic and structural properties of the native state of HisF. When considered with the results of previous studies where ILV clusters were found to stabilize the hydrogen-bonded networks in folding intermediates for other TIM barrel proteins, it appears that clusters of branched aliphatic side chains can serve as cores of stability across the entire folding reaction coordinate of one of the most common motifs in biology.