Robust prediction of relative binding energies for protein-protein complex mutations using free energy perturbation calculations.

Computational free energy-based methods have the potential to significantly improve throughput and decrease costs of protein design efforts. Such methods must reach a high level of reliability, accuracy, and automation to be effectively deployed in practical industrial settings in a way that impacts protein design projects. Here, we present a benchmark study for the calculation of relative changes in protein-protein binding affinity for single point mutations across a variety of systems from the literature, using free energy perturbation (FEP+) calculations. We describe a method for robust treatment of alternate protonation states for titratable amino acids, which yields improved correlation with and reduced error compared to experimental binding free energies. Following careful analysis of the largest outlier cases in our dataset, we assess limitations of the default FEP+ protocols and introduce an automated script which identifies probable outlier cases that may require additional scrutiny and calculates an empirical correction for a subset of charge-related outliers. Through a series of three additional case study systems, we discuss how protein FEP+ can be applied to real-world protein design projects, and suggest areas of further study.

Robust prediction of relative binding energies for protein-protein complex mutations using free energy perturbation calculations.

Computational free energy-based methods have the potential to significantly improve throughput and decrease costs of protein design efforts. Such methods must reach a high level of reliability, accuracy, and automation to be effectively deployed in practical industrial settings in a way that impacts protein design projects. Here, we present a benchmark study for the calculation of relative changes in protein-protein binding affinity for single point mutations across a variety of systems from the literature, using free energy perturbation (FEP+) calculations. We describe a method for robust treatment of alternate protonation states for titratable amino acids, which yields improved correlation with and reduced error compared to experimental binding free energies. Following careful analysis of the largest outlier cases in our dataset, we assess limitations of the default FEP+ protocols and introduce an automated script which identifies probable outlier cases that may require additional scrutiny and calculates an empirical correction for a subset of charge-related outliers. Through a series of three additional case study systems, we discuss how protein FEP+ can be applied to real-world protein design projects, and suggest areas of further study.

Free Energy Perturbation Calculations of Mutation Effects on SARS-CoV-2 RBD::ACE2 Binding Affinity.

The strength of binding between human angiotensin converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of viral spike protein plays a role in the transmissibility of the SARS-CoV-2 virus. In this study we focus on a subset of RBD mutations that have been frequently observed in infected individuals and probe binding affinity changes to ACE2 using surface plasmon resonance (SPR) measurements and free energy perturbation (FEP) calculations. Our SPR results are largely in accord with previous studies but discrepancies do arise due to differences in experimental methods and to protocol differences even when a single method is used. Overall, we find that FEP performance is superior to that of other computational approaches examined as determined by agreement with experiment and, in particular, by its ability to identify stabilizing mutations. Moreover, the calculations successfully predict the observed cooperative stabilization of binding by the Q498R N501Y double mutant present in Omicron variants and offer a physical explanation for the underlying mechanism. Overall, our results suggest that despite the significant computational cost, FEP calculations may offer an effective strategy to understand the effects of interfacial mutations on protein-protein binding affinities and, hence, in a variety of practical applications such as the optimization of neutralizing antibodies.

Affinity requirements for control of synaptic targeting and neuronal cell survival by heterophilic IgSF cell adhesion molecules.

Neurons in the developing brain express many different cell adhesion molecules (CAMs) on their surfaces. CAM-binding affinities can vary by more than 200-fold, but the significance of these variations is unknown. Interactions between the immunoglobulin superfamily CAM DIP-α and its binding partners, Dpr10 and Dpr6, control synaptic targeting and survival of Drosophila optic lobe neurons. We design mutations that systematically change interaction affinity and analyze function in vivo. Reducing affinity causes loss-of-function phenotypes whose severity scales with the magnitude of the change. Synaptic targeting is more sensitive to affinity reduction than is cell survival. Increasing affinity rescues neurons that would normally be culled by apoptosis. By manipulating CAM expression together with affinity, we show that the key parameter controlling circuit assembly is surface avidity, which is the strength of adherence between cell surfaces. We conclude that CAM binding affinities and expression levels are finely tuned for function during development.

Sorting of cadherin-catenin-associated proteins into individual clusters.

The cytoplasmic tails of classical cadherins form a multiprotein cadherin-catenin complex (CCC) that constitutes the major structural unit of adherens junctions (AJs). The CCC in AJs forms junctional clusters, "E clusters," driven by cis and trans interactions in the cadherin ectodomain and stabilized by α-catenin-actin interactions. Additional proteins are known to bind to the cytoplasmic region of the CCC. Here, we analyze how these CCC-associated proteins (CAPs) integrate into cadherin clusters and how they affect the clustering process. Using a cross-linking approach coupled with mass spectrometry, we found that the majority of CAPs, including the force-sensing protein vinculin, interact with CCCs outside of AJs. Accordingly, structural modeling shows that there is not enough space for CAPs the size of vinculin to integrate into E clusters. Using two CAPs, scribble and erbin, as examples, we provide evidence that these proteins form separate clusters, which we term "C clusters." As proof of principle, we show, by using cadherin ectodomain monoclonal antibodies (mAbs), that mAb-bound E-cadherin forms separate clusters that undergo trans interactions. Taken together, our data suggest that, in addition to its role in cell-cell adhesion, CAP-driven CCC clustering serves to organize cytoplasmic proteins into distinct domains that may synchronize signaling networks of neighboring cells within tissues.

DIP/Dpr interactions and the evolutionary design of specificity in protein families.

Differential binding affinities among closely related protein family members underlie many biological phenomena, including cell-cell recognition. Drosophila DIP and Dpr proteins mediate neuronal targeting in the fly through highly specific protein-protein interactions. We show here that DIPs/Dprs segregate into seven specificity subgroups defined by binding preferences between their DIP and Dpr members. We then describe a sequence-, structure- and energy-based computational approach, combined with experimental binding affinity measurements, to reveal how specificity is coded on the canonical DIP/Dpr interface. We show that binding specificity of DIP/Dpr subgroups is controlled by "negative constraints", which interfere with binding. To achieve specificity, each subgroup utilizes a different combination of negative constraints, which are broadly distributed and cover the majority of the protein-protein interface. We discuss the structural origins of negative constraints, and potential general implications for the evolutionary origins of binding specificity in multi-protein families.

Trans-endocytosis elicited by nectins transfers cytoplasmic cargo, including infectious material, between cells.

Here, we show that cells expressing the adherens junction protein nectin-1 capture nectin-4-containing membranes from the surface of adjacent cells in a trans-endocytosis process. We find that internalized nectin-1-nectin-4 complexes follow the endocytic pathway. The nectin-1 cytoplasmic tail controls transfer: its deletion prevents trans-endocytosis, while its exchange with the nectin-4 tail reverses transfer direction. Nectin-1-expressing cells acquire dye-labeled cytoplasmic proteins synchronously with nectin-4, a process most active during cell adhesion. Some cytoplasmic cargo remains functional after transfer, as demonstrated with encapsidated genomes of measles virus (MeV). This virus uses nectin-4, but not nectin-1, as a receptor. Epithelial cells expressing nectin-4, but not those expressing another MeV receptor in its place, can transfer infection to nectin-1-expressing primary neurons. Thus, this newly discovered process can move cytoplasmic cargo, including infectious material, from epithelial cells to neurons. We name the process nectin-elicited cytoplasm transfer (NECT). NECT-related trans-endocytosis processes may be exploited by pathogens to extend tropism. This article has an associated First Person interview with the first author of the paper.

Interactions between the Ig-Superfamily Proteins DIP-α and Dpr6/10 Regulate Assembly of Neural Circuits.

Drosophila Dpr (21 paralogs) and DIP proteins (11 paralogs) are cell recognition molecules of the immunoglobulin superfamily (IgSF) that form a complex protein interaction network. DIP and Dpr proteins are expressed in a synaptic layer-specific fashion in the visual system. How interactions between these proteins regulate layer-specific synaptic circuitry is not known. Here we establish that DIP-α and its interacting partners Dpr6 and Dpr10 regulate multiple processes, including arborization within layers, synapse number, layer specificity, and cell survival. We demonstrate that heterophilic binding between Dpr6/10 and DIP-α and homophilic binding between DIP-α proteins promote interactions between processes in vivo. Knockin mutants disrupting the DIP/Dpr binding interface reveal a role for these proteins during normal development, while ectopic expression studies support an instructive role for interactions between DIPs and Dprs in circuit development. These studies support an important role for the DIP/Dpr protein interaction network in regulating cell-type-specific connectivity patterns.

Neuron-Subtype-Specific Expression, Interaction Affinities, and Specificity Determinants of DIP/Dpr Cell Recognition Proteins.

Binding between DIP and Dpr neuronal recognition proteins has been proposed to regulate synaptic connections between lamina and medulla neurons in the Drosophila visual system. Each lamina neuron was previously shown to express many Dprs. Here, we demonstrate, by contrast, that their synaptic partners typically express one or two DIPs, with binding specificities matched to the lamina neuron-expressed Dprs. A deeper understanding of the molecular logic of DIP/Dpr interaction requires quantitative studies on the properties of these proteins. We thus generated a quantitative affinity-based DIP/Dpr interactome for all DIP/Dpr protein family members. This revealed a broad range of affinities and identified homophilic binding for some DIPs and some Dprs. These data, along with full-length ectodomain DIP/Dpr and DIP/DIP crystal structures, led to the identification of molecular determinants of DIP/Dpr specificity. This structural knowledge, along with a comprehensive set of quantitative binding affinities, provides new tools for functional studies in vivo.

Molecular basis of sidekick-mediated cell-cell adhesion and specificity.

Sidekick (Sdk) 1 and 2 are related immunoglobulin superfamily cell adhesion proteins required for appropriate synaptic connections between specific subtypes of retinal neurons. Sdks mediate cell-cell adhesion with homophilic specificity that underlies their neuronal targeting function. Here we report crystal structures of Sdk1 and Sdk2 ectodomain regions, revealing similar homodimers mediated by the four N-terminal immunoglobulin domains (Ig1-4), arranged in a horseshoe conformation. These Ig1-4 horseshoes interact in a novel back-to-back orientation in both homodimers through Ig1:Ig2, Ig1:Ig1 and Ig3:Ig4 interactions. Structure-guided mutagenesis results show that this canonical dimer is required for both Sdk-mediated cell aggregation (via trans interactions) and Sdk clustering in isolated cells (via cis interactions). Sdk1/Sdk2 recognition specificity is encoded across Ig1-4, with Ig1-2 conferring the majority of binding affinity and differential specificity. We suggest that competition between cis and trans interactions provides a novel mechanism to sharpen the specificity of cell-cell interactions.

α-Catenin-mediated cadherin clustering couples cadherin and actin dynamics.

The function of the actin-binding domain of α-catenin, αABD, including its possible role in the direct anchorage of the cadherin-catenin complex to the actin cytoskeleton, has remained uncertain. We identified two point mutations on the αABD surface that interfere with αABD binding to actin and used them to probe the role of α-catenin-actin interactions in adherens junctions. We found that the junctions directly bound to actin via αABD were more dynamic than the junctions bound to actin indirectly through vinculin and that recombinant αABD interacted with cortical actin but not with actin bundles. This interaction resulted in the formation of numerous short-lived cortex-bound αABD clusters. Our data suggest that αABD clustering drives the continuous assembly of transient, actin-associated cadherin-catenin clusters whose disassembly is maintained by actin depolymerization. It appears then that such actin-dependent αABD clustering is a unique molecular mechanism mediating both integrity and reassembly of the cell-cell adhesive interface formed through weak cis- and trans-intercadherin interactions.

Understanding boron through size-selected clusters: structure, chemical bonding, and fluxionality.

Boron is an interesting element with unusual polymorphism. While three-dimensional (3D) structural motifs are prevalent in bulk boron, atomic boron clusters are found to have planar or quasi-planar structures, stabilized by localized two-center-two-electron (2c-2e) σ bonds on the periphery and delocalized multicenter-two-electron (nc-2e) bonds in both σ and π frameworks. Electron delocalization is a result of boron's electron deficiency and leads to fluxional behavior, which has been observed in B13(+) and B19(-). A unique capability of the in-plane rotation of the inner atoms against the periphery of the cluster in a chosen direction by employing circularly polarized infrared radiation has been suggested. Such fluxional behaviors in boron clusters are interesting and have been proposed as molecular Wankel motors. The concepts of aromaticity and antiaromaticity have been extended beyond organic chemistry to planar boron clusters. The validity of these concepts in understanding the electronic structures of boron clusters is evident in the striking similarities of the π-systems of planar boron clusters to those of polycyclic aromatic hydrocarbons, such as benzene, naphthalene, coronene, anthracene, or phenanthrene. Chemical bonding models developed for boron clusters not only allowed the rationalization of the stability of boron clusters but also lead to the design of novel metal-centered boron wheels with a record-setting planar coordination number of 10. The unprecedented highly coordinated borometallic molecular wheels provide insights into the interactions between transition metals and boron and expand the frontier of boron chemistry. Another interesting feature discovered through cluster studies is boron transmutation. Even though it is well-known that B(-), formed by adding one electron to boron, is isoelectronic to carbon, cluster studies have considerably expanded the possibilities of new structures and new materials using the B(-)/C analogy. It is believed that the electronic transmutation concept will be effective and valuable in aiding the design of new boride materials with predictable properties. The study of boron clusters with intermediate properties between those of individual atoms and bulk solids has given rise to a unique opportunity to broaden the frontier of boron chemistry. Understanding boron clusters has spurred experimentalists and theoreticians to find new boron-based nanomaterials, such as boron fullerenes, nanotubes, two-dimensional boron, and new compounds containing boron clusters as building blocks. Here, a brief and timely overview is presented addressing the recent progress made on boron clusters and the approaches used in the authors' laboratories to determine the structure, stability, and chemical bonding of size-selected boron clusters by joint photoelectron spectroscopy and theoretical studies. Specifically, key findings on all-boron hydrocarbon analogues, metal-centered boron wheels, and electronic transmutation in boron clusters are summarized.

Theoretical study of the Si(5-n)(BH)n2- and Na(Si(5-n)(BH)n)- (n = 0-5) systems.

The potential energy surfaces of the Na(Si(5-n)(BH)(n))(-) and Si(5-n)(BH)(n)(2-) (n = 0-5) systems have been explored in detail. We established that all the global minimum structures of anionic and dianionic systems can be obtained by substitution of one or more silicon atoms of the global minima of NaSi(5)(-) and Si(5)(2-) for B-H units. The conservation of the overall structure upon isoelectronic substitution was shown to be due to the preservation of the chemical bonding pattern. Theoretical VDEs were calculated for all of the sodiated Na(Si(5-n)(BH)(n))(-) (n = 0-5) systems to facilitate their experimental detection.

B22- and B23-: all-boron analogues of anthracene and phenanthrene.

Clusters of boron atoms exhibit intriguing size-dependent structures and chemical bonding that are different from bulk boron and may lead to new boron-based nanostructures. We report a combined photoelectron spectroscopic and ab initio study of the 22- and 23-atom boron clusters. The joint experimental and theoretical investigation shows that B(22)(-) and B(23)(-) possess quasi-planar and planar structures, respectively. The quasi-planar B(22)(-) consists of fourteen peripheral atoms and eight interior atoms in a slightly buckled triangular lattice. Chemical bonding analyses of the closed-shell B(22)(2-) species reveal seven delocalized π orbitals, which are similar to those in anthracene. B(23)(-) is a perfectly planar and heart-shaped cluster with a pentagonal cavity and a π-bonding pattern similar to that in phenanthrene. Thus, B(22)(-) and B(23)(-), the largest negatively charged boron clusters that have been characterized experimentally to date, can be viewed as all-boron analogues of anthracene and phenanthrene, respectively. The current work shows not only that boron clusters are planar at very large sizes but also that they continue to yield surprises and novel chemical bonding analogous to specific polycyclic aromatic hydrocarbons.

B13+ : a photodriven molecular Wankel engine.

Revved-up rotary: A molecular Wankel motor, the dual-ring structure B(13)(+), is driven by circularly-polarized infrared electromagnetic radiation. Calculations show that this illumination leads to a guided unidirectional rotation of the outer ring, which is achieved with rotational frequency of the order of 300 GHz.

A photoelectron spectroscopy and ab initio study of B21-: negatively charged boron clusters continue to be planar at 21.

The structures and chemical bonding of the B(21)(-) cluster have been investigated by a combined photoelectron spectroscopy and ab initio study. The photoelectron spectrum at 193 nm revealed a very high adiabatic electron binding energy of 4.38 eV for B(21)(-) and a congested spectral pattern. Extensive global minimum searches were conducted using two different methods, followed by high-level calculations of the low-lying isomers. The global minimum of B(21)(-) was found to be a quasiplanar structure with the next low-lying planar isomer only 1.9 kcal/mol higher in energy at the CCSD(T)/6-311-G* level of theory. The calculated vertical detachment energies for the two isomers were found to be in good agreement with the experimental spectrum, suggesting that they were both present experimentally and contributed to the observed spectrum. Chemical bonding analyses showed that both isomers consist of a 14-atom periphery, which is bonded by classical two-center two-electron bonds, and seven interior atoms in the planar structures. A localized two-center two-electron bond is found in the interior of the two planar isomers, in addition to delocalized multi-center σ and π bonds. The structures and the delocalized bonding of the two lowest lying isomers of B(21)(-) were found to be similar to those in the two lowest energy isomers in B(19)(-).

All-boron analogues of aromatic hydrocarbons: B17- and B18-.

We have investigated the structural and electronic properties of the B(17)(-) and B(18)(-) clusters using photoelectron spectroscopy (PES) and ab initio calculations. The adiabatic electron detachment energies of B(17)(-) and B(18)(-) are measured to be 4.23 ± 0.02 and 3.53 ± 0.05 eV, respectively. Calculated electron detachment energies are compared with experimental data, confirming the presence of one planar C(2v) ((1)A(1)) isomer for B(17)(-) and two nearly isoenergetic quasi-planar C(3v) ((2)A(1)) and C(s) ((2)A') isomers for B(18)(-). The stability and planarity/quasi-planarity of B(17)(-) and B(18)(-) are ascribed to σ- and π-aromaticity. Chemical bonding analyses reveal that the nature of π-bonding in B(17)(-) and B(18)(-) is similar to that in the recently elucidated B(16)(2-) and B(19)(-) clusters, respectively. The planar B(17)(-) cluster can be considered as an all-boron analogue of naphthalene, whereas the π-bonding in the quasi-planar B(18)(-) is reminiscent of that in coronene.

Deciphering the mystery of hexagon holes in an all-boron graphene α-sheet.

Boron could be the next element after carbon capable of forming 2D-materials similar to graphene. Theoretical calculations predict that the most stable planar all-boron structure is the so-called α-sheet. The mysterious structure of the α-sheet with peculiar distribution of filled and empty hexagons is rationalized in terms of chemical bonding. We show that the hexagon holes serve as scavengers of extra electrons from the filled hexagons. This work could advance rational design of all-boron nanomaterials.

Unravelling phenomenon of internal rotation in B13+ through chemical bonding analysis.

We describe and explain the fluxionality of B(13)(+). The chemical bonding analysis shows that the inner triangle of B(13)(+) is bound to the peripheral ring by delocalized bonds only, allowing a quasi-free rotation of the inner ring.

Planarization of B7- and B12- clusters by isoelectronic substitution: AlB6- and AlB11-.

Small boron clusters have been shown to be planar from a series of combined photoelectron spectroscopy and theoretical studies. However, a number of boron clusters are quasiplanar, such as B(7)(-) and B(12)(-). To elucidate the nature of the nonplanarity in these clusters, we have investigated the electronic structure and chemical bonding of two isoelectronic Al-doped boron clusters, AlB(6)(-) and AlB(11)(-). Vibrationally resolved photoelectron spectra were obtained for AlB(6)(-), resulting in an accurate electron affinity (EA) for AlB(6) of 2.49 ± 0.03 eV. The photoelectron spectra of AlB(11)(-) revealed the presence of two isomers with EAs of 2.16 ± 0.03 and 2.33 ± 0.03 eV, respectively. Global minimum structures of both AlB(6)(-) and AlB(11)(-) were established from unbiased searches and comparison with the experimental data. The global minimum of AlB(6)(-) is nearly planar with a central B atom and an AlB(5) six membered ring, in contrast to that of B(7)(-), which possesses a C(2v) structure with a large distortion from planarity. Two nearly degenerate structures were found for AlB(11)(-) competing for the global minimum, in agreement with the experimental observation. One of these isomers with the lower EA can be viewed as substituting a peripheral B atom by Al in B(12)(-), which has a bowl shape with a B(9) outer ring and an out-of-plane inner B(3) triangle. The second isomer of AlB(11)(-) can be viewed as an Al atom interacting with a B(11)(-) cluster. Both isomers of AlB(11)(-) are perfectly planar. It is shown that Al substitution of a peripheral B atom in B(7)(-) and B(12)(-) induces planarization by slightly expanding the outer ring due to the larger size of Al.