Background:The mechanism of cataract formation by the recently discovered ␥D-crystallin W42R mutant is unknown. Results: Structural, biochemical, and biophysical studies revealed a partially unfolded species of the W42R mutant. Conclusion: Partially unfolded species serve as nuclei for aggregation. Significance: The properties of the W42R mutant ␥D-crystallin provide the link to the pathogenesis of age-related cataract caused by photodamaged wild-type ␥D-crystallin.

Although a number of γD-crystallin mutations are associated with cataract formation, there is not a clear understanding of the molecular mechanism(s) that lead to this protein deposition disease. As part of our ongoing studies on crystallins, we investigated the recently discovered Arg76 to Ser (R76S) mutation that is correlated with childhood cataract in an Indian family. We expressed the R76S γD-crystallin protein in E. coli, characterized it by CD, fluorescence, and NMR spectroscopy, and determined its stability with respect to thermal and chemical denaturation. Surprisingly, no significant biochemical or biophysical differences were observed between the wild-type protein and the R76S variant, except a lowered pI (6.8 compared to the wild-type value of 7.4). NMR assessment of the R76S γD-crystallin solution structure, by RDCs, and of its motional properties, by relaxation measurements, also revealed a close resemblance to wild type crystallin. Further, kinetic unfolding/refolding experiments for R76S and wild-type protein showed similar degrees of off-pathway aggregation suppression by αB-crystallin. Overall, our results suggest that neither structural nor stability changes in the protein are responsible for the R76S γD-crystallin variant's association with cataract. However, the change in pI and the associated surface charge or the altered nature of the amino acid could influence interactions with other lens protein species.

By means of limited proteolysis assay, three-dimensional NMR, X-ray crystallography and alanine mutations, a dynamic region at the Q221R222N223 motif in the Bcl-2 homology 3 (BH3) domain of Mcl-1 has been identified as a conformational switch which controls Mcl-1 ubiquitination. Noxa binding biases the QRN motif toward a helical conformation, thus leading to an enhanced in vitro ubiquitination of Mcl-1. In contrast, Bim binding biases the QRN motif toward a nonhelical conformation, thus leading to the inhibition of ubiquitination. A dual function Mcl-1 inhibitor, which locates at the BH3 domain of Mcl-1 and forms hydrogen bond with His224 to drive a helical QRN conformation, so that it not only interferes with the pro-apoptotic partners, but also facilitates Mcl-1 ubiquitination in living cells, is described. As a result, this inhibitor manifests a more effective apoptosis induction in Mcl-1-dependent cancer cells than other inhibitors exhibiting a similar binding affinity with it.

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Up to now, efforts to crystallize the cataract-associated P23T mutant of human γD-crystallin have not been successful. Therefore, insights into the light scattering mechanism of this mutant have been exclusively obtained from solution work. Here we present the first crystal structure of the P23T mutant at 2.5Å resolution. The protein exhibits essentially the same overall structure as seen for the wild-type protein. Based on our structural data, we confirm that no major conformational changes are caused by the mutation, and that solution phase properties of the mutant appear exclusively associated with cataract formation.

No abstract

By means of limited proteolysis assay, three‐dimensional NMR, X‐ray crystallography and alanine mutations, a dynamic region at the Q221R222N223 motif in the Bcl‐2 homology 3 (BH3) domain of Mcl‐1 has been identified as a conformational switch which controls Mcl‐1 ubiquitination. NoxaBH3 binding biases the QRN motif toward a helical conformation, thus leading to an enhanced in vitro ubiquitination of Mcl‐1. In contrast, BimBH3 binding biases the QRN motif toward a nonhelical conformation, thus leading to the inhibition of ubiquitination. A dual function Mcl‐1 inhibitor, which locates at the BH3 domain of Mcl‐1 and forms hydrogen bond with His224 to drive a helical QRN conformation, so that it not only interferes with the pro‐apoptotic partners, but also facilitates Mcl‐1 ubiquitination in living cells, is described. As a result, this inhibitor manifests a more effective apoptosis induction in Mcl‐1‐dependent cancer cells than other inhibitors exhibiting a similar binding affinity with it.

Acetoin is an important physiological metabolite excreted by microbes. Its functions include avoiding acidification, participating in regulation of the NAD/NADH ratio, and storing carbon. Acetolactate decarboxylase is a well-characterized anabolic enzyme involved with 3-hydroxy butanone (acetoin). It catalyzes conversion of the (R)- and (S)-enantiomers of acetolactate to generate the single product, (R)-acetoin. In addition to the X-ray crystal structure of acetolactate decarboxylase from Bacillus brevis, although the enzyme is widely present in microorganisms, very few atomic structures of acetolactate decarboxylase are reported. In this paper, we solved and reported a 1.5 Å resolution crystal structure of acetolactate decarboxylase from Bacillus subtilis. Dimeric assembly is observed in the solved structure, which is consistent with the elution profile conducted by molecular filtration. A zinc ion is coordinated by highly conserved histidines (191, 193, and 204) and conserved glutamates (62 and 251). We performed kinetic studies on acetolactate decarboxylase from Bacillus subtilis using circular dichroism, allowing the conversion of acetolactate to chiral acetoin for real-time tracking, yielding a K value of 21 mM and a k value of 2.2 s. Using the two enantiomers of acetolactate as substrates, we further investigated the substrate preference of acetolactate decarboxylase from Bacillus subtilis by means of molecular docking and dynamic simulation in silico. The binding free energy of (S)-acetolactate was found to be ~ 30 kcal/mol greater than that of (R)-acetolactate, indicating a more stable binding for (S)-acetolactate.