Reaction network starting from monomer mixtures of Aβ40 and Aβ42. Interaction at the level of primary nucleation only accelerates Aβ40 fibril formation. Separate fibrils form as secondary nucleation and elongation are highly specific.

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The recognition of carbohydrates by proteins is a fundamental aspect of communication within and between living cells. Understanding the molecular basis of carbohydrate–protein interactions is a prerequisite for the rational design of synthetic ligands. Here we report the high- to ultra-high-resolution crystal structures of the carbohydrate recognition domain of galectin-3 (Gal3C) in the ligand-free state (1.08 Å at 100 K, 1.25 Å at 298 K) and in complex with lactose (0.86 Å) or glycerol (0.9 Å). These structures reveal striking similarities in the positions of water and carbohydrate oxygen atoms in all three states, indicating that the binding site of Gal3C is preorganized to coordinate oxygen atoms in an arrangement that is nearly optimal for the recognition of β-galactosides. Deuterium nuclear magnetic resonance (NMR) relaxation dispersion experiments and molecular dynamics simulations demonstrate that all water molecules in the lactose-binding site exchange with bulk water on a time scale of nanoseconds or shorter. Nevertheless, molecular dynamics simulations identify transient water binding at sites that agree well with those observed by crystallography, indicating that the energy landscape of the binding site is maintained in solution. All heavy atoms of glycerol are positioned like the corresponding atoms of lactose in the Gal3C complexes. However, binding of glycerol to Gal3C is insignificant in solution at room temperature, as monitored by NMR spectroscopy or isothermal titration calorimetry under conditions where lactose binding is readily detected. These observations make a case for protein cryo-crystallography as a valuable screening method in fragment-based drug discovery and further suggest that identification of water sites might inform inhibitor design.

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Cold shock proteins (CSP) belong to the family of single-stranded nucleic acid binding proteins with OB-fold. CSP are believed to function as 'RNA chaperones' and during anti-termination. We determined the solution structure of Bs-CspB bound to the single-stranded DNA (ssDNA) fragment heptathymidine (dT 7 ) by NMR spectroscopy. BsCspB reveals an almost invariant conformation when bound to dT 7 with only minor reorientations in loop b1-b2 and b3-b4 and of few aromatic side chains involved in base stacking. Binding studies of protein variants and mutated ssDNA demonstrated that Bs-CspB associates with ssDNA at almost diffusion controlled rates and low sequence specificity consistent with its biological function. A variation of the ssDNA affinity is accomplished solely by changes of the dissociation rate. 15 N NMR relaxation and H/D exchange experiments revealed that binding of dT 7 increases the stability of Bs-CspB and reduces the sub-nanosecond dynamics of the entire protein and especially of loop b3-b4.

Intramolecular motions of proteins are critical for biological function. Transient structural fluctuations underlie a wide range of processes, including enzyme catalysis, ligand binding to buried sites, and generic protein motions, such as 180° rotation of aromatic side chains in the protein interior, but remain poorly understood. Understanding the dynamics and molecular nature of concerted motions requires characterization of their rates and energy barriers. Here we use recently developed (13)C transverse relaxation dispersion methods to improve our current understanding of aromatic ring flips in basic pancreatic trypsin inhibitor (BPTI). We validate these methods by benchmarking ring-flip rates against the three previously characterized cases in BPTI, namely, Y23, Y35, and F45. Further, we measure conformational exchange for one additional aromatic ring, F22, which can be interpreted in terms of a flip rate of 666 s(-1) at 5 °C. Upon inclusion of our previously reported result that Y21 also flips slowly [Weininger, U., et al. (2013) J. Phys. Chem. B 117, 9241-9247], the (13)C relaxation dispersion experiments thus reveal relatively slow ring-flip rates for five of eight aromatic residues in BPTI. These results are in contrast with previous reports, which have estimated that all rings, except Y23, Y35, and F45, flip with a high rate at ambient temperature. The (13)C relaxation dispersion data result in an updated rank order of ring-flip rates in BPTI, which agrees considerably better with that estimated from a recent 1 ms molecular dynamics trajectory than do previously published NMR data. However, significant quantitative differences remain between experiment and simulation, in that the latter yields flip rates that are in many cases too fast by 1-2 orders of magnitude. By measuring flip rates across a temperature range of 5-65 °C, we determined the activation barriers of ring flips for Y23, Y35, and F45. Y23 and F45 have identical activation parameters, suggesting that the fluctuations of the protein core around these residues are similar in character. Y35 differs from the other two in its apparent activation entropy. These results might be rationalized by the fact that Y23 and F45 are located in the same region of the structure while Y35 is remote from the other two rings. As indicated by our new results for the exceptionally well-characterized protein BPTI, (13)C relaxation dispersion experiments open the possibility of studying ring flips in a range of cases wider than that previously possible.

The human molecular chaperone DNAJB6, an oligomeric protein with a conserved S/T-rich region, is an efficient suppressor of amyloid fibril formation by highly aggregation-prone peptides such as the Aβ and polyQ peptides associated with Alzheimer's and Huntington's disease, respectively. We previously showed that DNAJB6 can inhibit the processes through which amyloid fibrils are formed via strong interactions with aggregated forms of Aβ42 that become sequestered. Here we report that the concentration-dependent capability of DNAJB6 to suppress fibril formation in thioflavin T fluorescence assays decreases progressively with an increasing number of S/T substitutions, with an almost complete loss of suppression when 18 S/T residues are substituted. The kinetics of primary nucleation in particular are affected. No detectable changes in the structure are caused by the substitutions. Also, the level of binding of DNAJB6 to Aβ42 decreases with the S/T substitutions, as determined by surface plasmon resonance and microscale thermophoresis. The aggregation process monitored using nuclear magnetic resonance spectroscopy showed that DNAJB6, in contrast to a mutational variant with 18 S/T residues substituted, can keep monomeric Aβ42 soluble for an extended time. The inhibition of the primary nucleation is likely to depend on hydroxyl groups in side chains of the S/T residues, and hydrogen bonding with Aβ42 is one plausible molecular mechanism, although other possibilities cannot be excluded. The loss of the ability to suppress fibril formation upon S/T to A substitution was previously observed also for polyQ peptides, suggesting that the S/T residues in the DNAJB6-like chaperones have a general ability to inhibit amyloid fibril formation by different aggregation-prone peptides.

Allosteric communication within proteins is a hallmark of biochemical signaling, but the dynamic transmission pathways remain poorly characterized. We combined NMR spectroscopy and surface plasmon resonance to reveal these pathways and quantify their energetics in the glucocorticoid receptor, a transcriptional regulator controlling development, metabolism, and immune response. Our results delineate a dynamic communication network of residues linking the ligand-binding pocket to the activation function-2 interface, where helix 12, a switch for transcriptional activation, exhibits ligand- and coregulator-dependent dynamics coupled to graded activation. The allosteric free energy responds to variations in ligand structure: subtle changes gradually tune allostery while preserving the transmission pathway, whereas substitution of the entire pharmacophore leads to divergent allosteric control by apparently rewiring the communication network. Our results provide key insights that should aid in the design of mechanistically differentiated ligands.