Machine learning differentiates enzymatic and non-enzymatic metals in proteins

Metalloenzymes are 40% of all enzymes and can perform all seven classes of enzyme reactions. Because of the physicochemical similarities between the active sites of metalloenzymes and inactive metal binding sites, it is challenging to differentiate between them. Yet distinguishing these two classes is critical for the identification of both native and designed enzymes. Because of similarities between catalytic and non-catalytic metal binding sites, finding physicochemical features that distinguish these two types of metal sites can indicate aspects that are critical to enzyme function. In this work, we develop the largest structural dataset of enzymatic and non-enzymatic metalloprotein sites to date. We then use a decision-tree ensemble machine learning model to classify metals bound to proteins as enzymatic or non-enzymatic with 92.2% precision and 90.1% recall. Our model scores electrostatic and pocket lining features as more important than pocket volume, despite the fact that volume is the most quantitatively different feature between enzyme and non-enzymatic sites. Finally, we find our model has overall better performance in a side-to-side comparison against other methods that differentiate enzymatic from non-enzymatic sequences. We anticipate that our model’s ability to correctly identify which metal sites are responsible for enzymatic activity could enable identification of new enzymatic mechanisms and de novo enzyme design

Efflux Pumps Represent Possible Evolutionary Convergence onto the Beta Barrel Fold

Preprint now published in Structure doi: 10.1016/j.str.2018.06.007There are around 100 types of integral outer membrane proteins in each Gram negative bacteria. All of these proteins have the same fold—an up-down β-barrel. It has been suggested that all membrane β-barrels other than lysins are homologous. Here we suggest that β-barrels of efflux pumps have converged on this fold as well. By grouping structurally-solved outer membrane β-barrels (OMBBs) by sequence we find evidence that the membrane environment may have led to convergent evolution of the barrel fold. Specifically, the lack of sequence linkage to other barrels coupled with distinctive structural differences, such as differences in strand tilt and barrel radius, suggest that efflux pumps have evolutionarily converged on the barrel. Finally, we find a possible ancestor for the OMBB efflux pumps as they are related to periplasmic components of the same pumps

Antiparallel Dimers of the Small Multidrug Resistance Protein EmrE Are More Stable Than Parallel Dimers

The bacterial multidrug transporter EmrE is a dual-topology membrane protein and as such is able to insert into the membrane in two opposite orientations. The functional form of EmrE is a homodimer; however, the relative orientation of the subunits in the dimer is under debate. Using EmrE variants with fixed, opposite orientations in the membrane, we now show that, although the proteins are able to form parallel dimers, an antiparallel organization of the subunits in the dimer is preferred. Blue-native PAGE analyses of intact oligomers and disulfide cross-linking demonstrate that in membranes, the proteins form parallel dimers only if no oppositely orientated partner is present. Co-expression of oppositely orientated proteins almost exclusively yields antiparallel dimers. Finally, parallel dimers can be disrupted and converted into antiparallel dimers by heating of detergent-solubilized protein. Importantly, in vivo function is correlated clearly to the presence of antiparallel dimers. Our results suggest that an antiparallel arrangement of the subunits in the dimer is more stable than a parallel organization and likely corresponds to the functional form of the protein

Modulating Integrin αIIbβ3 Activity through Mutagenesis of Allosterically Regulated Intersubunit Contacts

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Biochemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.biochem.9b00430.Integrin αIIbβ3, a transmembrane heterodimer, mediates platelet aggregation when it switches from an inactive to an active ligand-binding conformation following platelet stimulation. Central to regulating αIIbβ3 activity is the interaction between the αIIb and β3 extracellular stalks, which form a tight heterodimer in the inactive state and dissociate in the active state. Here, we demonstrate that alanine replacements of sensitive positions in the heterodimer stalk interface destabilize the inactive conformation sufficiently to cause constitutive αIIbβ3 activation. To determine the structural basis for this effect, we performed a structural bioinformatics analysis and found that perturbing intersubunit contacts with favorable interaction geometry through substitutions to alanine quantitatively accounted for the degree of constitutive αIIbβ3 activation. This mutational study directly assesses the relationship between favorable interaction geometry at mutation-sensitive positions and the functional activity of those mutants, giving rise to a simple model that highlights the importance of interaction geometry in contributing to the stability between protein–protein interactions.NIH P01 HL40387NIH R35 GM122603National Science Foundation 1709506National Science Foundation 165011

Sketching nature\u27s energy function: Designing helices that target transmembrane proteins and creating software for further design

A variety of methods exist for the design or selection of antibodies and other proteins that recognize the water-soluble regions of proteins, however companion methods for targeting transmembrane (TM) regions are not available. Here, I describe two methods for the computational design of peptides that target TM helices in a sequence-specific manner—one for the design of native-like L-peptides, and one for the design of D-peptides. To illustrate these methods, peptides were designed that specifically recognize the TM helices of two closely related integrins, αIIbβ3 and αvβ3, in micelles, bacterial membranes, and mammalian cells. These data show that sequence-specific recognition of helices in TM proteins can be achieved through optimization of the geometric complementarity of the target/host complex. I have also developed new design software to further explore forces that make proteins fold and to create more proteins that can probe biological function

Sketching nature\u27s energy function: Designing helices that target transmembrane proteins and creating software for further design

A variety of methods exist for the design or selection of antibodies and other proteins that recognize the water-soluble regions of proteins, however companion methods for targeting transmembrane (TM) regions are not available. Here, I describe two methods for the computational design of peptides that target TM helices in a sequence-specific manner—one for the design of native-like L-peptides, and one for the design of D-peptides. To illustrate these methods, peptides were designed that specifically recognize the TM helices of two closely related integrins, αIIbβ3 and αvβ3, in micelles, bacterial membranes, and mammalian cells. These data show that sequence-specific recognition of helices in TM proteins can be achieved through optimization of the geometric complementarity of the target/host complex. I have also developed new design software to further explore forces that make proteins fold and to create more proteins that can probe biological function