Gevorg Grigoryan

Design of protein-interaction specificity gives selective bZIP-binding peptides

Interaction specificity is a required feature of biological networks and a necessary characteristic of protein or small-molecule reagents and therapeutics. The ability to alter or inhibit protein interactions selectively would advance basic and applied molecular science. Assessing or modelling interaction specificity requires treating multiple competing complexes, which presents computational and experimental challenges. Here we present a computational framework for designing protein-interaction specificity and use it to identify specific peptide partners for human basic-region leucine zipper (bZIP) transcription factors. Protein microarrays were used to characterize designed, synthetic ligands for all but one of 20 bZIP families. The bZIP proteins share strong sequence and structural similarities and thus are challenging targets to bind specifically. Nevertheless, many of the designs, including examples that bind the oncoproteins c-Jun, c-Fos and c-Maf (also called JUN, FOS and MAF, respectively), were selective for their targets over all 19 other families. Collectively, the designs exhibit a wide range of interaction profiles and demonstrate that human bZIPs have only sparsely sampled the possible interaction space accessible to them. Our computational method provides a way to systematically analyse trade-offs between stability and specificity and is suitable for use with many types of structure-scoring functions; thus, it may prove broadly useful as a tool for protein design.

Structural specificity in coiled-coil interactions

Coiled coils have a rich history in the field of protein design and engineering. Novel structures, such as the first seven-helix coiled coil, continue to provide surprises and insights. Large-scale datasets quantifying the influence of systematic mutations on coiled-coil stability are a valuable new asset to the area. Scoring methods based on sequence and/or structure can predict interaction preferences in coiled-coil-mediated bZIP transcription factor dimerization. Experimental and computational methods for dealing with the near-degeneracy of many coiled-coil structures appear promising for future design applications.

Computational Design of Virus-like Protein Assemblies on Carbon Nanotube Surfaces

Proteins are designed to bind to specific surfaces while also presenting a programmed surface superstructure. There is a general need for the engineering of protein-like molecules that organize into geometrically specific superstructures on molecular surfaces, directing further functionalization to create richly textured, multilayered assemblies. Here we describe a computational approach whereby the surface properties and symmetry of a targeted surface define the sequence and superstructure of surface-organizing peptides. Computational design proceeds in a series of steps that encode both surface recognition and favorable intersubunit packing interactions. This procedure is exemplified in the design of peptides that assemble into a tubular structure surrounding single-walled carbon nanotubes (SWNTs). The geometrically defined, virus-like coating created by these peptides converts the smooth surfaces of SWNTs into highly textured assemblies with long-scale order, capable of directing the assembly of gold nanoparticles into helical arrays along the SWNT axis.

De novo design of a transmembrane Zn2+-transporting four-helix bundle.

The design of functional membrane proteins from first principles represents a grand challenge in chemistry and structural biology. Here, we report the design of a membrane-spanning, four-helical bundle that transports first-row transition metal ions Zn2+ and Co2+, but not Ca2+, across membranes. The conduction path was designed to contain two di-metal binding sites that bind with negative cooperativity. X-ray crystallography and solid-state and solution nuclear magnetic resonance indicate that the overall helical bundle is formed from two tightly interacting pairs of helices, which form individual domains that interact weakly along a more dynamic interface. Vesicle flux experiments show that as Zn2+ ions diffuse down their concentration gradients, protons are antiported. These experiments illustrate the feasibility of designing membrane proteins with predefined structural and dynamic properties. Computational design yields a transmembrane protein that selectively transports zinc cations. [Also see Perspective by Lupas] Building transmembrane zinc transporters The ability to design proteins gives insight into the relation between a proteins fold and its function and also provides a path to custom proteins for bioengineering applications. Impressive strides have been made in the design of soluble proteins, but designing membrane proteins remains a challenge. Joh et al. achieve a milestone by designing a transmembrane Zn2+ transporter (see the Perspective by Lupas). The protein comprises four helices: Two tightly interacting pairs form a weaker interface that facilitates the transport of Zn2+ with concomitant reverse transport of protons. Science, this issue p. 1520; see also p. 1455

Point-based probabilistic surfaces to show surface uncertainty

Efficient and informative visualization of surfaces with uncertainties is an important topic with many applications in science and engineering. In these applications, the correct course of action may depend not only on the location of a boundary, but on the precision with which that location is known. Examples include environmental pollution borderline detection, oil basin edge characterization, or discrimination between cancerous and healthy tissue in medicine. We present a method for producing visualizations of surfaces with uncertainties using points as display primitives. Our approach is to render the surface as a collection of points and to displace each point from its original location along the surface normal by an amount proportional to the uncertainty at that point. This approach can be used in combination with other techniques such as pseudocoloring to produce efficient and revealing visualizations. The basic approach is sufficiently flexible to allow natural extensions; we show incorporation of expressive modulation of opacity, change of the stroke primitive, and addition of an underlying polygonal model. The method is used to visualize real and simulated tumor formations with uncertainty of tumor boundaries. The point-based technique is compared to pseudocoloring for a position estimation task in a preliminary user study.

Capture and imaging of a prehairpin fusion intermediate of the paramyxovirus PIV5

During cell entry, enveloped viruses fuse their viral membrane with a cellular membrane in a process driven by energetically favorable, large-scale conformational rearrangements of their fusion proteins. Structures of the pre- and postfusion states of the fusion proteins including paramyxovirus PIV5 F and influenza virus hemagglutinin suggest that this occurs via two intermediates. Following formation of an initial complex, the proteins structurally elongate, driving a hydrophobic N-terminal “fusion peptide” away from the protein surface into the target membrane. Paradoxically, this first conformation change moves the viral and cellular bilayers further apart. Next, the fusion proteins form a hairpin that drives the two membranes into close opposition. While the pre- and postfusion hairpin forms have been characterized crystallographically, the transiently extended prehairpin intermediate has not been visualized. To provide evidence for this extended intermediate we measured the interbilayer spacing of a paramyxovirus trapped in the process of fusing with solid-supported bilayers. A gold-labeled peptide that binds the prehairpin intermediate was used to stabilize and specifically image F-proteins in the prehairpin intermediate. The interbilayer spacing is precisely that predicted from a computational model of the prehairpin, providing strong evidence for its structure and functional role. Moreover, the F-proteins in the prehairpin conformation preferentially localize to a patch between the target and viral membranes, consistent with the fact that the formation of the prehairpin is triggered by local contacts between F- and neighboring viral receptor-binding proteins (HN) only when HN binds lipids in its target membrane.

Identification of bZIP interaction partners of viral proteins HBZ, MEQ, BZLF1, and K-bZIP using coiled-coil arrays.

Basic-region leucine-zipper transcription factors (bZIPs) contain a segment rich in basic amino acids that can bind DNA, followed by a leucine zipper that can interact with other leucine zippers to form coiled-coil homo- or heterodimers. Several viruses encode proteins containing bZIP domains, including four that encode bZIPs lacking significant homology to any human protein. We investigated the interaction specificity of these four viral bZIPs by using coiled-coil arrays to assess self-associations as well as heterointeractions with 33 representative human bZIPs. The arrays recapitulated reported viral-human interactions and also uncovered new associations. MEQ and HBZ interacted with multiple human partners and had unique interaction profiles compared to any human bZIPs, whereas K-bZIP and BZLF1 displayed homospecificity. New interactions detected included HBZ with MAFB, MAFG, ATF2, CEBPG, and CREBZF and MEQ with NFIL3. These were confirmed in solution using circular dichroism. HBZ can heteroassociate with MAFB and MAFG in the presence of MARE-site DNA, and this interaction is dependent on the basic region of HBZ. NFIL3 and MEQ have different yet overlapping DNA-binding specificities and can form a heterocomplex with DNA. Computational design considering both affinity for MEQ and specificity with respect to other undesired bZIP-type interactions was used to generate a MEQ dimerization inhibitor. This peptide, anti-MEQ, bound MEQ both stably and specifically, as assayed using coiled-coil arrays and circular dichroism in solution. Anti-MEQ also inhibited MEQ binding to DNA. These studies can guide further investigation of the function of viral and human bZIP complexes.

Ultra-Fast Evaluation of Protein Energies Directly from Sequence

The structure, function, stability, and many other properties of a protein in a fixed environment are fully specified by its sequence, but in a manner that is difficult to discern. We present a general approach for rapidly mapping sequences directly to their energies on a pre-specified rigid backbone, an important sub-problem in computational protein design and in some methods for protein structure prediction. The cluster expansion (CE) method that we employ can, in principle, be extended to model any computable or measurable protein property directly as a function of sequence. Here we show how CE can be applied to the problem of computational protein design, and use it to derive excellent approximations of physical potentials. The approach provides several attractive advantages. First, following a one-time derivation of a CE expansion, the amount of time necessary to evaluate the energy of a sequence adopting a specified backbone conformation is reduced by a factor of 107 compared to standard full-atom methods for the same task. Second, the agreement between two full-atom methods that we tested and their CE sequence-based expressions is very high (root mean square deviation 1.1–4.7 kcal/mol, R2 = 0.7–1.0). Third, the functional form of the CE energy expression is such that individual terms of the expansion have clear physical interpretations. We derived expressions for the energies of three classic protein design targets—a coiled coil, a zinc finger, and a WW domain—as functions of sequence, and examined the most significant terms. Single-residue and residue-pair interactions are sufficient to accurately capture the energetics of the dimeric coiled coil, whereas higher-order contributions are important for the two more globular folds. For the task of designing novel zinc-finger sequences, a CE-derived energy function provides significantly better solutions than a standard design protocol, in comparable computation time. Given these advantages, CE is likely to find many uses in computational structural modeling.

Probabilistic surfaces: point based primitives to show surface uncertainty

Efficient and informative visualization of surfaces with uncertainties is an important topic with many applications in science and engineering. Examples include environmental pollution borderline identification, identification of the limits of an oil basin, or discrimination between contaminated and healthy tissue in medicine. This paper presents an approach for such visualization using points as display primitives. The approach is to render each polygon as a collection of points and to displace each point from the surface in the direction of the surface normal by an amount proportional to some random number multiplied by the uncertainty level at that point. This approach can be used in combination with other techniques such as pseudo-coloring and shading to give rise to efficient and revealing visualizations. The method is used to visualize real and simulated tumor formations with uncertainty of tumor boundaries.

Transmembrane Communication: General Principles and Lessons from the Structure and Function of the M2 Proton Channel, K+ Channels, and Integrin Receptors

Signal transduction across biological membranes is central to life. This process generally happens through communication between different domains and hierarchical coupling of information. Here, we review structural and thermodynamic principles behind transmembrane (TM) signal transduction and discuss common themes. Communication between signaling domains can be understood in terms of thermodynamic and kinetic principles, and complex signaling patterns can arise from simple wiring of thermodynamically coupled domains. We relate this to functions of several signal transduction systems: the M2 proton channel from influenza A virus, potassium channels, integrin receptors, and bacterial kinases. We also discuss key features in the structural rearrangements responsible for signal transduction in these systems.