Cold Spot SCANNER: Colab Notebook for predicting cold spots in protein-protein interfaces.

BACKGROUND: Protein-protein interactions (PPIs) are conveyed through binding interfaces or surface patches on proteins that become buried upon binding. Structural and biophysical analysis of many protein-protein interfaces revealed certain unique features of these surfaces that determine the energetics of interactions and play a critical role in protein evolution. One of the significant aspects of binding interfaces is the presence of binding hot spots, where mutations are highly deleterious for binding. Conversely, binding cold spots are positions occupied by suboptimal amino acids and several mutations in such positions could lead to affinity enhancement. While there are many software programs for identification of hot spot positions, there is currently a lack of software for cold spot detection. RESULTS: In this paper, we present Cold Spot SCANNER, a Colab Notebook, which scans a PPI binding interface and identifies cold spots resulting from cavities, unfavorable charge-charge, and unfavorable charge-hydrophobic interactions. The software offers a Py3DMOL-based interface that allows users to visualize cold spots in the context of the protein structure and generates a zip file containing the results for easy download. CONCLUSIONS: Cold spot identification is of great importance to protein engineering studies and provides a useful insight into protein evolution. Cold Spot SCANNER is open to all users without login requirements and can be accessible at: https://colab. RESEARCH: google.com/github/sagagugit/Cold-Spot-Scanner/blob/main/Cold_Spot_Scanner.ipynb .

Mirror-Image Random Nonstandard Peptide Integrated Discovery (MI-RaPID) Technology Yields Highly Stable and Selective Macrocyclic Peptide Inhibitors for Matrix Metallopeptidase 7.

Matrix metallopeptidase 7 (MMP7) plays a crucial role in cancer metastasis and progression, making it an attractive target for therapeutic development. However, the development of selective MMP7 inhibitors is challenging due to the conservation of active sites across various matrix metalloproteinases (MMPs). Here, we have developed mirror-image random nonstandard peptides integrated discovery (MI-RaPID) technology to discover innate protease-resistant macrocyclic peptides that specifically bind to and inhibit human MMP7. One identified macrocyclic peptide against D-MMP7, termed D20, was synthesized in its mirror-image form, D'20, consisting of 12 D-amino acids, one cyclic b-amino acid, and a thioether bond. Notably, it potently inhibited MMP7 with an IC50 value of 90 nM, and showed excellent selectivity over other MMPs with similar substrate specificity. Moreover, D'20 inhibited the migration of pancreatic cell line CFPAC-1, but had no effect on the cell proliferation and viability. D'20 exhibited excellent stability in human serum, as well as in simulated gastric and intestinal fluids. This study highlights that MI-RaPID technology can serve as a powerful tool to develop in vivo stable macrocyclic peptides for therapeutic applications.

Engineered variants of the Ras effector protein RASSF5 (NORE1A) promote anticancer activities in lung adenocarcinoma.

Within the superfamily of small GTPases, Ras appears to be the master regulator of such processes as cell cycle progression, cell division, and apoptosis. Several oncogenic Ras mutations at amino acid positions 12, 13, and 61 have been identified that lose their ability to hydrolyze GTP, giving rise to constitutive signaling and eventually development of cancer. While disruption of the Ras/effector interface is an attractive strategy for drug design to prevent this constitutive activity, inhibition of this interaction using small molecules is impractical due to the absence of a cavity to which such molecules could bind. However, proteins and especially natural Ras effectors that bind to the Ras/effector interface with high affinity could disrupt Ras/effector interactions and abolish procancer pathways initiated by Ras oncogene. Using a combination of computational design and in vitro evolution, we engineered high-affinity Ras-binding proteins starting from a natural Ras effector, RASSF5 (NORE1A), which is encoded by a tumor suppressor gene. Unlike previously reported Ras oncogene inhibitors, the proteins we designed not only inhibit Ras-regulated procancer pathways, but also stimulate anticancer pathways initiated by RASSF5. We show that upon introduction into A549 lung carcinoma cells, the engineered RASSF5 mutants decreased cell viability and mobility to a significantly greater extent than WT RASSF5. In addition, these mutant proteins induce cellular senescence by increasing acetylation and decreasing phosphorylation of p53. In conclusion, engineered RASSF5 variants provide an attractive therapeutic strategy able to oppose cancer development by means of inhibiting of procancer pathways and stimulating anticancer processes.

Climbing Up and Down Binding Landscapes through Deep Mutational Scanning of Three Homologous Protein-Protein Complexes.

Protein-protein interactions (PPIs) have evolved to display binding affinities that can support their function. As such, cognate and noncognate PPIs could be highly similar structurally but exhibit huge differences in binding affinities. To understand this phenomenon, we study three homologous protease-inhibitor PPIs that span 9 orders of magnitude in binding affinity. Using state-of-the-art methodology that combines protein randomization, affinity sorting, deep sequencing, and data normalization, we report quantitative binding landscapes consisting of ΔΔGbind values for the three PPIs, gleaned from tens of thousands of single and double mutations. We show that binding landscapes of the three complexes are strikingly different and depend on the PPI evolutionary optimality. We observe different patterns of couplings between mutations for the three PPIs with negative and positive epistasis appearing most frequently at hot-spot and cold-spot positions, respectively. The evolutionary trends observed here are likely to be universal to other biological complexes in the cell.

Cold Spots in Protein Binding.

Understanding the energetics and architecture of protein-binding interfaces is important for basic research and could potentially facilitate the design of novel binding domains for biotechnological applications. It is well accepted that a few key residues at binding interfaces (binding hot spots) are responsible for contributing most to the free energy of binding. In this opinion article, we introduce a new concept of 'binding cold spots', or interface positions occupied by suboptimal amino acids. Such positions exhibit a potential for affinity enhancement through various mutations. We give several examples of cold spots from different protein-engineering studies and argue that identification of such positions is crucial for studies of protein evolution and protein design.

Protein Engineering by Combined Computational and In Vitro Evolution Approaches.

Two alternative strategies are commonly used to study protein-protein interactions (PPIs) and to engineer protein-based inhibitors. In one approach, binders are selected experimentally from combinatorial libraries of protein mutants that are displayed on a cell surface. In the other approach, computational modeling is used to explore an astronomically large number of protein sequences to select a small number of sequences for experimental testing. While both approaches have some limitations, their combination produces superior results in various protein engineering applications. Such applications include the design of novel binders and inhibitors, the enhancement of affinity and specificity, and the mapping of binding epitopes. The combination of these approaches also aids in the understanding of the specificity profiles of various PPIs.

Saturation scanning of ubiquitin variants reveals a common hot spot for binding to USP2 and USP21.

A detailed understanding of the molecular mechanisms whereby ubiquitin (Ub) recognizes enzymes in the Ub proteasome system is crucial for understanding the biological function of Ub. Many structures of Ub complexes have been solved and, in most cases, reveal a large structural epitope on a common face of the Ub molecule. However, owing to the generally weak nature of these interactions, it has been difficult to map in detail the functional contributions of individual Ub side chains to affinity and specificity. Here we took advantage of Ub variants (Ubvs) that bind tightly to particular Ub-specific proteases (USPs) and used phage display and saturation scanning mutagenesis to comprehensively map functional epitopes within the structural epitopes. We found that Ubvs that bind to USP2 or USP21 contain a remarkably similar core functional epitope, or "hot spot," consisting mainly of positions that are conserved as the wild type sequence, but also some positions that prefer mutant sequences. The Ubv core functional epitope contacts residues that are conserved in the human USP family, and thus it is likely important for the interactions of Ub across many family members.

Development of High Affinity and High Specificity Inhibitors of Matrix Metalloproteinase 14 through Computational Design and Directed Evolution.

Degradation of the extracellular matrices in the human body is controlled by matrix metalloproteinases (MMPs), a family of more than 20 homologous enzymes. Imbalance in MMP activity can result in many diseases, such as arthritis, cardiovascular diseases, neurological disorders, fibrosis, and cancers. Thus, MMPs present attractive targets for drug design and have been a focus for inhibitor design for as long as 3 decades. Yet, to date, all MMP inhibitors have failed in clinical trials because of their broad activity against numerous MMP family members and the serious side effects of the proposed treatment. In this study, we integrated a computational method and a yeast surface display technique to obtain highly specific inhibitors of MMP-14 by modifying the natural non-specific broad MMP inhibitor protein N-TIMP2 to interact optimally with MMP-14. We identified an N-TIMP2 mutant, with five mutations in its interface, that has an MMP-14 inhibition constant (Ki ) of 0.9 pm, the strongest MMP-14 inhibitor reported so far. Compared with wild-type N-TIMP2, this variant displays ∼900-fold improved affinity toward MMP-14 and up to 16,000-fold greater specificity toward MMP-14 relative to other MMPs. In an in vitro and cell-based model of MMP-dependent breast cancer cellular invasiveness, this N-TIMP2 mutant acted as a functional inhibitor. Thus, our study demonstrates the enormous potential of a combined computational/directed evolution approach to protein engineering. Furthermore, it offers fundamental clues into the molecular basis of MMP regulation by N-TIMP2 and identifies a promising MMP-14 inhibitor as a starting point for the development of protein-based anticancer therapeutics.

Combinatorial and Computational Approaches to Identify Interactions of Macrophage Colony-stimulating Factor (M-CSF) and Its Receptor c-FMS.

The molecular interactions between macrophage colony-stimulating factor (M-CSF) and the tyrosine kinase receptor c-FMS play a key role in the immune response, bone metabolism, and the development of some cancers. Because no x-ray structure is available for the human M-CSF · c-FMS complex, the binding epitope for this complex is largely unknown. Our goal was to identify the residues that are essential for binding of the human M-CSF to c-FMS. For this purpose, we used a yeast surface display (YSD) approach. We expressed a combinatorial library of monomeric M-CSF (M-CSFM) single mutants and screened this library to isolate variants with reduced affinity for c-FMS using FACS. Sequencing yielded a number of single M-CSFM variants with mutations both in the direct binding interface and distant from the binding site. In addition, we used computational modeling to map the identified mutations onto the M-CSFM structure and to classify the mutations into three groups as follows: those that significantly decrease protein stability; those that destroy favorable intermolecular interactions; and those that decrease affinity through allosteric effects. To validate the YSD and computational data, M-CSFM and three variants were produced as soluble proteins; their affinity and structure were analyzed; and very good correlations with both YSD data and computational predictions were obtained. By identifying the M-CSFM residues critical for M-CSF · c-FMS interactions, we have laid down the basis for a deeper understanding of the M-CSF · c-FMS signaling mechanism and for the development of target-specific therapeutic agents with the ability to sterically occlude the M-CSF·c-FMS binding interface.

Synthetic peptides mimicking the binding site of human acetylcholinesterase for its inhibitor fasciculin 2.

Molecules capable of mimicking protein binding and/or functional sites present useful tools for a range of biomedical applications, including the inhibition of protein-ligand interactions. Such mimics of protein binding sites can currently be generated through structure-based design and chemical synthesis. Computational protein design could be further used to optimize protein binding site mimetics through rationally designed mutations that improve intermolecular interactions or peptide stability. Here, as a model for the study, we chose an interaction between human acetylcholinesterase (hAChE) and its inhibitor fasciculin-2 (Fas) because the structure and function of this complex is well understood. Structure-based design of mimics of the hAChE binding site for Fas yielded a peptide that binds to Fas at micromolar concentrations. Replacement of hAChE residues known to be essential for its interaction with Fas with alanine, in this peptide, resulted in almost complete loss of binding to Fas. Computational optimization of the hAChE mimetic peptide yielded a variant with slightly improved affinity to Fas, indicating that more rounds of computational optimization will be required to obtain peptide variants with greatly improved affinity for Fas. CD spectra in the absence and presence of Fas point to conformational changes in the peptide upon binding to Fas. Furthermore, binding of the optimized hAChE mimetic peptide to Fas could be inhibited by hAChE, providing evidence for a hAChE-specific peptide-Fas interaction.

ProBASS - a language model with sequence and structural features for predicting the effect of mutations on binding affinity.

Protein-protein interactions (PPIs) govern virtually all cellular processes. Even a single mutation within PPI can significantly influence overall protein functionality and potentially lead to various types of diseases. To date, numerous approaches have emerged for predicting the change in free energy of binding (ΔΔGbind) resulting from mutations, yet the majority of these methods lack precision. In recent years, protein language models (PLMs) have been developed and shown powerful predictive capabilities by leveraging both sequence and structural data from protein-protein complexes. Yet, PLMs have not been optimized specifically for predicting ΔΔGbind. We developed an approach to predict effects of mutations on PPI binding affinity based on two most advanced protein language models ESM2 and ESM-IF1 that incorporate PPI sequence and structural features, respectively. We used the two models to generate embeddings for each PPI mutant and subsequently fine-tuned our model by training on a large dataset of experimental ΔΔGbind values. Our model, ProBASS (Protein Binding Affinity from Structure and Sequence) achieved a correlation with experimental ΔΔGbind values of 0.83 ± 0.05 for single mutations and 0.69 ± 0.04 for double mutations when model training and testing was done on the same PDB. Moreover, ProBASS exhibited very high correlation (0.81 ± 0.02) between prediction and experiment when training and testing was performed on a dataset containing 2325 single mutations in 132 PPIs. ProBASS surpasses the state-of-the-art methods in correlation with experimental data and could be further trained as more experimental data becomes available. Our results demonstrate that the integration of extensive datasets containing ΔΔGbind values across multiple PPIs to refine the pre-trained PLMs represents a successful approach for achieving a precise and broadly applicable model for ΔΔGbind prediction, greatly facilitating future protein engineering and design studies.

Improving Circulation Half-Life of Therapeutic Candidate N-TIMP2 by Unfolded Peptide Extension.

Matrix metalloproteinases (MMPs) are significant drivers of many diseases, including cancer, and are established targets for drug development. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous MMP inhibitors and are being pursued for the development of anti-MMP therapeutics. TIMPs possess many attractive properties for drug candidates, such as complete MMP inhibition, low toxicity, low immunogenicity, and high tissue permeability. However, a major challenge with TIMPs is their rapid clearance from the bloodstream due to their small size. This study explores a method for extending the plasma half-life of the N-terminal domain of TIMP2 (N-TIMP2) by appending it with a long, intrinsically unfolded tail containing Pro, Ala, and Thr (PATylation). We designed and produced two PATylated N-TIMP2 constructs with tail lengths of 100 and 200 amino acids (N-TIMP2-PAT100 and N-TIMP2-PAT200). Both constructs demonstrated higher apparent molecular weights and retained high inhibitory activity against MMP-9. N-TIMP2-PAT200 significantly increased plasma half-life in mice compared to the non-PATylated variant, enhancing its therapeutic potential. PATylation offers distinct advantages for half-life extension, such as fully genetic encoding, monodispersion, and biodegradability. It can be easily applied to N-TIMP2 variants engineered for high affinity and selectivity toward individual MMPs, creating promising candidates for drug development against MMP-related diseases.

Improving Circulation Half-Life of Therapeutic Candidate N-TIMP2 by Unfolded Peptide Extension.

Matrix Metalloproteinases (MMPs) are drivers of many diseases including cancer and are established targets for drug development. Tissue inhibitors of metalloproteinases (TIMPs) are human proteins that inhibit MMPs and are being pursued for the development of anti-MMP therapeutics. TIMPs possess many attractive properties of a drug candidate, such as complete MMP inhibition, low toxicity and immunogenicity, high tissue permeability and others. A major challenge with TIMPs, however, is their formulation and delivery, as these proteins are quickly cleared from the bloodstream due to their small size. In this study, we explore a new method for plasma half-life extension for the N-terminal domain of TIMP2 (N-TIMP2) through appending it with a long intrinsically unfolded tail containing a random combination of Pro, Ala, and Thr (PATylation). We design, produce and explore two PATylated N-TIMP2 constructs with a tail length of 100- and 200-amino acids (N-TIMP2-PAT100 and N-TIMP2-PAT200, respectively). We demonstrate that both PATylated N-TIMP2 constructs possess apparent higher molecular weights compared to the wild-type protein and retain high inhibitory activity against MMP-9. Furthermore, when injected into mice, N-TIMP2-PAT200 exhibited a significant increase in plasma half-life compared to the non-PATylated variant, enhancing the therapeutic potential of the protein. Thus, we establish that PATylation could be successfully applied to TIMP-based therapeutics and offers distinct advantages as an approach for half-life extension, such as fully genetic encoding of the gene construct, mono-dispersion, and biodegradability. Furthermore, PATylation could be easily applied to N-TIMP2 variants engineered to possess high affinity and selectivity toward individual MMP family members, thus creating attractive candidates for drug development against MMP-related diseases.

Predicting affinity- and specificity-enhancing mutations at protein-protein interfaces.

Manipulations of PPIs (protein-protein interactions) are important for many biological applications such as synthetic biology and drug design. Combinatorial methods have been traditionally used for such manipulations, failing, however, to explain the effects achieved. We developed a computational method for prediction of changes in free energy of binding due to mutation that bring about deeper understanding of the molecular forces underlying binding interactions. Our method could be used for computational scanning of binding interfaces and subsequent analysis of the interfacial sequence optimality. The computational method was validated in two biological systems. Computational saturated mutagenesis of a high-affinity complex between an enzyme AChE (acetylcholinesterase) and a snake toxin Fas (fasciculin) revealed the optimal nature of this interface with only a few predicted affinity-enhancing mutations. Binding measurements confirmed high optimality of this interface and identified a few mutations that could further improve interaction fitness. Computational interface scanning of a medium-affinity complex between TIMP-2 (tissue inhibitor of metalloproteinases-2) and MMP (matrix metalloproteinase) 14 revealed a non-optimal nature of the binding interface with multiple mutations predicted to stabilize the complex. Experimental results corroborated our computational predictions, identifying a large number of mutations that improve the binding affinity for this interaction and some mutations that enhance binding specificity. Overall, our computational protocol greatly facilitates the discovery of affinity- and specificity-enhancing mutations and thus could be applied for design of potent and highly specific inhibitors of any PPI.

Targeting Ras with protein engineering.

Ras proteins are small GTPases that regulate cell growth and division. Mutations in Ras genes are associated with many types of cancer, making them attractive targets for cancer therapy. Despite extensive efforts, targeting Ras proteins with small molecules has been extremely challenging due to Ras's mostly flat surface and lack of small molecule-binding cavities. These challenges were recently overcome by the development of the first covalent small-molecule anti-Ras drug, sotorasib, highlighting the efficacy of Ras inhibition as a therapeutic strategy. However, this drug exclusively inhibits the Ras G12C mutant, which is not a prevalent mutation in most cancer types. Unlike the G12C variant, other Ras oncogenic mutants lack reactive cysteines, rendering them unsuitable for targeting via the same strategy. Protein engineering has emerged as a promising method to target Ras, as engineered proteins have the ability to recognize various surfaces with high affinity and specificity. Over the past few years, scientists have engineered antibodies, natural Ras effectors, and novel binding domains to bind to Ras and counteract its carcinogenic activities via a variety of strategies. These include inhibiting Ras-effector interactions, disrupting Ras dimerization, interrupting Ras nucleotide exchange, stimulating Ras interaction with tumor suppressor genes, and promoting Ras degradation. In parallel, significant advancements have been made in intracellular protein delivery, enabling the delivery of the engineered anti-Ras agents into the cellular cytoplasm. These advances offer a promising path for targeting Ras proteins and other challenging drug targets, opening up new opportunities for drug discovery and development.

Designed Loop Extension Followed by Combinatorial Screening Confers High Specificity to a Broad Matrix MetalloproteinaseInhibitor.

Matrix metalloproteinases (MMPs) are key drivers of various diseases, including cancer. Development of probes and drugs capable of selectively inhibiting the individual members of the large MMP family remains a persistent challenge. The inhibitory N-terminal domain of tissue inhibitor of metalloproteinases-2 (N-TIMP2), a natural broad MMP inhibitor, can provide a scaffold for protein engineering to create more selective MMP inhibitors. Here, we pursued a unique approach harnessing both computational design and combinatorial screening to confer high binding specificity toward a target MMP in preference to an anti-target MMP. We designed a loop extension of N-TIMP2 to allow new interactions with the non-conserved MMP surface and generated an efficient focused library for yeast surface display, which was then screened for high binding to the target MMP-14 and low binding to anti-target MMP-3. Deep sequencing analysis identified the most promising variants, which were expressed, purified, and tested for selectivity of inhibition. Our best N-TIMP2 variant exhibited 29 pM binding affinity to MMP-14 and 2.4 µM affinity to MMP-3, revealing 7500-fold greater specificity than WT N-TIMP2. High-confidence structural models were obtained by including NGS data in the AlphaFold multiple sequence alignment. The modeling together with experimental mutagenesis validated our design predictions, demonstrating that the loop extension packs tightly against non-conserved residues on MMP-14 and clashes with MMP-3. This study demonstrates how introduction of loop extensions in a manner guided by target protein conservation data and loop design can offer an attractive strategy to achieve specificity in design of protein ligands.

Cold spots are universal in protein-protein interactions.

Proteins interact with each other through binding interfaces that differ greatly in size and physico-chemical properties. Within the binding interface, a few residues called hot spots contribute the majority of the binding free energy and are hence irreplaceable. In contrast, cold spots are occupied by suboptimal amino acids, providing possibility for affinity enhancement through mutations. In this study, we identify cold spots due to cavities and unfavorable charge interactions in multiple protein-protein interactions (PPIs). For our cold spot analysis, we first use a small affinity database of PPIs with known structures and affinities and then expand our search to nearly 4000 homo- and heterodimers in the Protein Data Bank (PDB). We observe that cold spots due to cavities are present in nearly all PPIs unrelated to their binding affinity, while unfavorable charge interactions are relatively rare. We also find that most cold spots are located in the periphery of the binding interface, with high-affinity complexes showing fewer centrally located colds spots than low-affinity complexes. A larger number of cold spots is also found in non-cognate interactions compared to their cognate counterparts. Furthermore, our analysis reveals that cold spots are more frequent in homo-dimeric complexes compared to hetero-complexes, likely due to symmetry constraints imposed on sequences of homodimers. Finally, we find that glycines, glutamates, and arginines are the most frequent amino acids appearing at cold spot positions. Our analysis emphasizes the importance of cold spot positions to protein evolution and facilitates protein engineering studies directed at enhancing binding affinity and specificity in a wide range of applications.

Multispecific recognition: mechanism, evolution, and design.

Accumulating evidence shows that many particular proteins have evolved to bind multiple targets, including other proteins, peptides, DNA, and small molecule substrates. Multispecific recognition might be not only common but also necessary for the robustness of signaling and metabolic networks in the cell. It is also important for the immune response and for regulation of transcription and translation. Multispecificity presents an apparent paradox: How can a protein encoded by a single sequence accommodate numerous targets? Analysis of sequences and structures of multispecific proteins revealed a number of mechanisms that achieve multispecificity. Interestingly, similar mechanisms appear in antibody-antigen, T-cell receptor-peptide, protein-DNA, enzyme-substrate, and protein-protein complexes. Directed evolution and protein design experiments with multispecific proteins offer some interesting insights into the evolution of such proteins and help in the dissection of molecular interactions that mediate multispecificity. Understanding the basic principles governing multispecificity could greatly assist in the unraveling of various complex processes in the cell. In addition, through manipulation of functional multispecificity, novel proteins could be created for use in various biotechnological and biomedical applications.

Transfer-PCR (TPCR): a highway for DNA cloning and protein engineering.

DNA cloning and protein engineering are basic methodologies employed for various applications in all life-science disciplines. Manipulations of DNA however, could be a lengthy process that slows down subsequent experiments. To facilitate both DNA cloning and protein engineering, we present Transfer-PCR (TPCR), a novel approach that integrates in a single tube, PCR amplification of the target DNA from an origin vector and its subsequent integration into the destination vector. TPCR can be applied for incorporation of DNA fragments into any desired position within a circular plasmid without the need for purification of the intermediate PCR product and without the use of any commercial kit. Using several examples, we demonstrate the applicability of the TPCR platform for both DNA cloning and for multiple-site targeted mutagenesis. In both cases, we show that the TPCR reaction is most efficient within a narrow range of primer concentrations. In mutagenesis, TPCR is primarily advantageous for generation of combinatorial libraries of targeted mutants but could be also applied to generation of variants with specific multiple mutations throughout the target gene. Adaptation of the TPCR platform should facilitate, simplify and significantly reduce time and costs for diverse protein structure and functional studies.

Triathlon for energy functions: who is the winner for design of protein-protein interactions?

Computational prediction of stabilizing mutations into monomeric proteins has become an almost ordinary task. Yet, computational stabilization of protein­protein complexes remains a challenge. Design of protein­protein interactions (PPIs) is impeded by the absence of an energy function that could reliably reproduce all favorable interactions between the binding partners. In this work, we present three energy functions: one function that was trained on monomeric proteins, while the other two were optimized by different techniques to predict side-chain conformations in a dataset of PPIs. The performances of these energy functions are evaluated in three different tasks related to design of PPIs: predicting side-chain conformations in PPIs, recovering native binding-interface sequences, and predicting changes in free energy of binding due to mutations. Our findings show that both functions optimized on side-chain repacking in PPIs are more suitable for PPI design compared to the function trained on monomeric proteins. Yet, no function performs best at all three tasks. Comparison of the three energy functions and their performances revealed that (1) burial of polar atoms should not be penalized significantly in PPI design as in single-protein design and (2) contribution of electrostatic interactions should be increased several-fold when switching from single-protein to PPI design. In addition, the use of a softer van der Waals potential is beneficial in cases when backbone flexibility is important. All things considered, we define an energy function that captures most of the nuances of the binding energetics and hence, should be used in future for design of PPIs.