Elusive affinities

The affinity of two molecules for each other and its temperature dependence are determined by the change in enthalpy, free enthalpy, entropy, and heat capacity upon dissociation. As we know the forces that stabilize-protein–protein or protein–DNA association and the three-dimensional structures of the complex, we can in principle derive values for each one of these parameters. The calculation is done first in gas phase by molecular mechanics, then in solution with the help of hydration parameters calibrated on small molecules. However, estimates of enthalpy and entropy changes in gas phase have excessively large error bars even under the approximation that the components of the complex associate as rigid bodies. No reliable result can be expected at the end. The fit to experimental values derived from binding and calorimetric measurements is poor, except for the dissociation heat capacity. This parameter can be attributed mostly to the hydration step and it correlates with the size of the interface. Many protein–protein complexes have interface areas in the range 1200–2000 Ų and only small conformation changes, so the rigid body approximation applies. It is less generally valid in protein–DNA complexes, which have interfaces covering 2200–3100 Ų, large dissociation heat capacities, and affect both the conformation and the dynamics of their components. © 1995 Wiley-Liss, Inc.     

Molecular architecture of LSM14 interactions involved in the assembly of mRNA silencing complexes

The LSM domain‐containing protein LSM14/Rap55 plays a role in mRNA decapping, translational repression, and RNA granule (P‐body) assembly. How LSM14 interacts with the mRNA silencing machinery, including the eIF4E‐binding protein 4E‐T and the DEAD‐box helicase DDX6, is poorly understood. Here we report the crystal structure of the LSM domain of LSM14 bound to a highly conserved C‐terminal fragment of 4E‐T. The 4E‐T C‐terminus forms a bi‐partite motif that wraps around the N‐terminal LSM domain of LSM14. We also determined the crystal structure of LSM14 bound to the C‐terminal RecA‐like domain of DDX6. LSM14 binds DDX6 via a unique non‐contiguous motif with distinct directionality as compared to other DDX6‐interacting proteins. Together with mutational and proteomic studies, the LSM14‐DDX6 structure reveals that LSM14 has adopted a divergent mode of binding DDX6 in order to support the formation of mRNA silencing complexes and P‐body assembly.     

Mitotic spindle association of TACC3 requires Aurora‐A‐dependent stabilization of a cryptic α‐helix

Aurora‐A regulates the recruitment of TACC3 to the mitotic spindle through a phospho‐dependent interaction with clathrin heavy chain (CHC). Here, we describe the structural basis of these interactions, mediated by three motifs in a disordered region of TACC3. A hydrophobic docking motif binds to a previously uncharacterized pocket on Aurora‐A that is blocked in most kinases. Abrogation of the docking motif causes a delay in late mitosis, consistent with the cellular distribution of Aurora‐A complexes. Phosphorylation of Ser558 engages a conformational switch in a second motif from a disordered state, needed to bind the kinase active site, into a helical conformation. The helix extends into a third, adjacent motif that is recognized by a helical‐repeat region of CHC, not a recognized phospho‐reader domain. This potentially widespread mechanism of phospho‐recognition provides greater flexibility to tune the molecular details of the interaction than canonical recognition motifs that are dominated by phosphate binding.     

Subunit-specific backbone NMR assignments of a 64 kDa trp repressor/DNA complex: A role for N-terminal residues in tandem binding

Deuterium decoupled, triple resonance NMR spectroscopy was used to analyze complexes of ²H,¹⁵N,¹³C labelled intact and (des2–7) trp repressor (2–7 trpR) from E. coli bound in tandem to an idealized 22 basepair trp operator DNA fragment and the corepressor 5-methyltryptophan. The DNA sequence used here binds two trpR dimers in tandem resulting in chemically nonequivalent environments for the two subunits of each dimer. Sequence- and subunit-specific NMR resonance assignments were made for backbone ¹HN, ¹⁵N, ¹³C positions in both forms of the protein and for¹³ C in the intact repressor. The differences in backbone chemical shifts between the two subunits within each dimer of 2–7 trpR reflect dimer-dimer contacts involving the helix-turn-helix domains and N-terminal residues consistent with a previously determined crystal structure [Lawson and Carey (1993) Nature, 366, 178–182]. Comparison of the backbone chemical shifts of DNA-bound 2–7 trpR with those of DNA-bound intact trpR reveals significant changes for those residues involved in N-terminal-mediated interactions observed in the crystal structure. In addition, our solution NMR data contain three sets of resonances for residues 2–12 in intact trpR suggesting that the N-terminus has multiple conformations in the tandem complex. Analysis of C chemical shifts using a chemical shift index (CSI) modified for deuterium isotope effects has allowed a comparison of the secondary structure of intact and 2–7 tprR. Overall these data demonstrate that NMR backbone chemical shift data can be readily used to study specific structural details of large protein complexes.