Comparison of a molecular dynamics model with the X-ray structure of the N370S acid-beta-glucosidase mutant that causes Gaucher disease.

Recently, two studies were published that examined the structure of the acid-ß-glucosidase N370S mutant, the most common mutant that causes Gaucher disease. One study used the experimental tool of X-ray crystallography, and the other utilized molecular dynamics (MD). The two studies reinforced each other through the similarities in their findings, but each approach also added some unique information. Both studies report that the conformation of active site loop 3 changes, due to an altered hydrogen bonding network; however, the MD study produced additional data concerning the flexibility of loop 1 and the catalytic residues that are not observed in the other study.

A mutation in VPS35, encoding a subunit of the retromer complex, causes late-onset Parkinson disease.

To identify rare causal variants in late-onset Parkinson disease (PD), we investigated an Austrian family with 16 affected individuals by exome sequencing. We found a missense mutation, c.1858G>A (p.Asp620Asn), in the VPS35 gene in all seven affected family members who are alive. By screening additional PD cases, we saw the same variant cosegregating with the disease in an autosomal-dominant mode with high but incomplete penetrance in two further families with five and ten affected members, respectively. The mean age of onset in the affected individuals was 53 years. Genotyping showed that the shared haplotype extends across 65 kilobases around VPS35. Screening the entire VPS35 coding sequence in an additional 860 cases and 1014 controls revealed six further nonsynonymous missense variants. Three were only present in cases, two were only present in controls, and one was present in cases and controls. The familial mutation p.Asp620Asn and a further variant, c.1570C>T (p.Arg524Trp), detected in a sporadic PD case were predicted to be damaging by sequence-based and molecular-dynamics analyses. VPS35 is a component of the retromer complex and mediates retrograde transport between endosomes and the trans-Golgi network, and it has recently been found to be involved in Alzheimer disease.

Rational engineering of L-asparaginase reveals importance of dual activity for cancer cell toxicity.

Using proteins in a therapeutic context often requires engineering to modify functionality and enhance efficacy. We have previously reported that the therapeutic antileukemic protein macromolecule Escherichia coli L-asparaginase is degraded by leukemic lysosomal cysteine proteases. In the present study, we successfully engineered L-asparaginase to resist proteolytic cleavage and at the same time improve activity. We employed a novel combination of mutant sampling using a genetic algorithm in tandem with flexibility studies using molecular dynamics to investigate the impact of lid-loop and mutations on drug activity. Applying these methods, we successfully predicted the more active L-asparaginase mutants N24T and N24A. For the latter, a unique hydrogen bond network contributes to higher activity. Furthermore, interface mutations controlling secondary glutaminase activity demonstrated the importance of this enzymatic activity for drug cytotoxicity. All selected mutants were expressed, purified, and tested for activity and for their ability to form the active tetrameric form. By introducing the N24A and N24A R195S mutations to the drug L-asparaginase, we are a step closer to individualized drug design.

Molecular basis of reduced glucosylceramidase activity in the most common Gaucher disease mutant, N370S.

Gaucher disease is caused by the defective activity of the lysosomal hydrolase, glucosylceramidase. Although the x-ray structure of wild type glucosylceramidase has been resolved, little is known about the structural features of any of the >200 mutations. Various treatments for Gaucher disease are available, including enzyme replacement and chaperone therapies. The latter involves binding of competitive inhibitors at the active site to enable correct folding and transport of the mutant enzyme to the lysosome. We now use molecular dynamics, a set of structural analysis tools, and several statistical methods to determine the flexible behavior of the N370S Gaucher mutant at various pH values, with and without binding the chaperone, N-butyl-deoxynojirimycin. We focus on the effect of the chaperone on the whole protein, on the active site, and on three important structural loops, and we demonstrate how the chaperone modifies the behavior of N370S in such a way that it becomes more active at lysosomal pH. Our results suggest a mechanism whereby the binding of N-butyl-deoxynojirimycin helps target correctly folded glucosylceramidase to the lysosome, contributes to binding with saposin C, and explains the initiation of the substrate-enzyme complex. Such analysis provides a new framework for determination of the structure of other Gaucher disease mutants and suggests new approaches for rational drug design.

A dyad of lymphoblastic lysosomal cysteine proteases degrades the antileukemic drug L-asparaginase.

l-Asparaginase is a key therapeutic agent for treatment of childhood acute lymphoblastic leukemia (ALL). There is wide individual variation in pharmacokinetics, and little is known about its metabolism. The mechanisms of therapeutic failure with l-asparaginase remain speculative. Here, we now report that 2 lysosomal cysteine proteases present in lymphoblasts are able to degrade l-asparaginase. Cathepsin B (CTSB), which is produced constitutively by normal and leukemic cells, degraded asparaginase produced by Escherichia coli (ASNase) and Erwinia chrysanthemi. Asparaginyl endopeptidase (AEP), which is overexpressed predominantly in high-risk subsets of ALL, specifically degraded ASNase. AEP thereby destroys ASNase activity and may also potentiate antigen processing, leading to allergic reactions. Using AEP-mediated cleavage sequences, we modeled the effects of the protease on ASNase and created a number of recombinant ASNase products. The N24 residue on the flexible active loop was identified as the primary AEP cleavage site. Sole modification at this site rendered ASNase resistant to AEP cleavage and suggested a key role for the flexible active loop in determining ASNase activity. We therefore propose what we believe to be a novel mechanism of drug resistance to ASNase. Our results may help to identify alternative therapeutic strategies with the potential of further improving outcome in childhood ALL.

Alternating evolutionary pressure in a genetic algorithm facilitates protein model selection.

BACKGROUND: Automatic protein modelling pipelines are becoming ever more accurate; this has come hand in hand with an increasingly complicated interplay between all components involved. Nevertheless, there are still potential improvements to be made in template selection, refinement and protein model selection. RESULTS: In the context of an automatic modelling pipeline, we analysed each step separately, revealing several non-intuitive trends and explored a new strategy for protein conformation sampling using Genetic Algorithms (GA). We apply the concept of alternating evolutionary pressure (AEP), i.e. intermediate rounds within the GA runs where unrestrained, linear growth of the model populations is allowed. CONCLUSION: This approach improves the overall performance of the GA by allowing models to overcome local energy barriers. AEP enabled the selection of the best models in 40% of all targets; compared to 25% for a normal GA.

Developing a move-set for protein model refinement.

MOTIVATION: A wide variety of methods for the construction of an atomic model for a given amino acid sequence are known, the more accurate being those that use experimentally determined structures as templates. However, far fewer methods are aimed at refining these models. The approach presented here carefully blends models created by several different means, in an attempt to combine the good quality regions from each into a final, more refined, model. RESULTS: We describe here a number of refinement operators (collectively, 'move-set') that enable a relatively large region of conformational space to be searched. This is used within a genetic algorithm that reshuffles and repacks structural components. The utility of the move-set is demonstrated by introducing a cost function, containing both physical and other components guiding the input structures towards the target structure. We show that our move-set has the potential to improve the conformation of models and that this improvement can be beyond even the best template for some comparative modelling targets. AVAILABILITY: The populus software package and the source code are available at http://bmm.cancerresearchuk.org/~offman01/populus.html.

No statistical support for correlation between the positions of protein interaction sites and alternatively spliced regions.

BACKGROUND: Alternative splicing is an efficient mechanism for increasing the variety of functions fulfilled by proteins in a living cell. It has been previously demonstrated that alternatively spliced regions often comprise functionally important and conserved sequence motifs. The objective of this work was to test the hypothesis that alternative splicing is correlated with contact regions of protein-protein interactions. RESULTS: Protein sequence spans involved in contacts with an interaction partner were delineated from atomic structures of transient interaction complexes and juxtaposed with the location of alternatively spliced regions detected by comparative genome analysis and spliced alignment. The total of 42 alternatively spliced isoforms were identified in 21 amino acid chains involved in biomolecular interactions. Using this limited dataset and a variety of sophisticated counting procedures we were not able to establish a statistically significant correlation between the positions of protein interaction sites and alternatively spliced regions. CONCLUSIONS: This finding contradicts a naïve hypothesis that alternatively spliced regions would correlate with points of contact. One possible explanation for that could be that all alternative splicing events change the spatial structure of the interacting domain to a sufficient degree to preclude interaction. This is indirectly supported by the observed lack of difference in the behaviour of relatively short regions affected by alternative splicing and cases when large portions of proteins are removed. More structural data on complexes of interacting proteins, including structures of alternative isoforms, are needed to test this conjecture.