Mitogen‐activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less‐characterized disordered regions. We used a structurally consistent model on kinase‐docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under‐explored part of the human proteome and applied experimental tools specifically tailored to detect low‐affinity protein–protein interactions for their validation in vitro and in cell‐based assays. The combined computational and experimental approach enabled the identification of many novel MAPK‐docking motifs that were elusive for other large‐scale protein–protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase‐mediated partnerships evolved over time. The analysis suggests that most human MAPK‐binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK‐binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles.

Despite a large number of antiretroviral drugs targeting HIV-1 protease for inhibition, mutations in this protein during the course of patient treatment can render them inefficient. This emerging resistance inspired numerous computational studies of the HIV-1 protease aimed at predicting the effect of mutations on drug binding in terms of free binding energy ΔG, as well as in mechanistic terms. In this study, we analyze ten different protease-inhibitor complexes carrying major resistance-associated mutations (RAMs) G48V, I50V, and L90M using molecular dynamics simulations. We demonstrate that alchemical free energy calculations can consistently predict the effect of mutations on drug binding. By explicitly probing different protonation states of the catalytic aspartic dyad, we reveal the importance of the correct choice of protonation state for the accuracy of the result. We also provide insight into how different mutations affect drug binding in their specific ways, with the unifying theme of how all of them affect the crucial drug binding regions of the protease. ■ INTRODUCTIONHIV-1 (human immunodeficiency virus-1, further denoted as HIV) has caused a global epidemic that affects approximately 37 million people worldwide.1 There is no vaccine or cure available against HIV, but antiretroviral therapy (ART) is recommended for every infected individual 1 to suppress the virus. Success of ART has led to a near-normal life expectancy of HIV infected patients.2 Nevertheless, resistance toward drugs remains a major issue, making it necessary to switch therapy during the course of treatment of a single patient. 1One of the main targets of ART is the HIV protease, a protein responsible for cleaving HIV polyproteins during virus maturation ( Figure 1a). HIV protease is a homodimer with two 99 residues long subunits. The major structural components of the binding pocket of this protein are the active site at its bottom, which is followed in sequence by a loop and a short β-sheet (residues 26−32) that constitute the side of the pocket together with the so-called 80s loop (residues 79−84) ( Figure 1a). On the top the pocket is covered by the so-called flap region. In the framework of ART, HIV protease can be inhibited by protease inhibitors (PIs) (Figure 1b), a class of competitive inhibitors which are currently recommended as second-and third-line ART treatment.1 Specific substitutions in 13 different positions in the protease are considered to be major resistanceassociated mutations (RAMs). 3I50V is a RAM toward protease inhibitors Lopinavir (LPV) 4,5 and Darunavir (DRV) 6,7 and has been associated with particularly strong resistance to Amprenavir (APV) 8−10 and correspondingly to its prodrug Fosamprenavir (FPV). It also has been suggested to cause resistance toward Indinavir (IDV), 9,11 and at the same time, there is evidence pointing that this mutation has been associated with sensitivity toward Atazanavir (ATV) 12,13 and Tipranavir (TPV).14 I50 is located in the protease flap region (Figure 1a). This flap is...

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