Structural characteristics of protein binding sites for calcium and lanthanide ions

Surveys of X-ray structures of Ca2+-containing and lanthanide ion-containing proteins and coordination complexes have been performed and structural features of the metal binding sites compared. A total of 515 structures of Ca2+-containing proteins were considered, although the final data set contained only 44 structures and 60 Ca2+ binding sites with a total of 323 ligands. Eighteen protein structures containing lanthanide ions were considered with a final data set containing eight structures and 11 metal binding sites. Structural features analysed include coordination numbers of the metal ions, the identity of their ligands, the denticity of carboxylate ligands, and the type of secondary structure from which the ligands are derived. Three general types of calcium binding site were identified in the final data set: class I sites supply the Ca2+ ligands from a continuous short sequence of amino acids; class II sites have one ligand supplied by a part of the amino acid sequence far removed from the main binding sequence; and class III sites are created by amino acids remote from one another in the sequence. The abundant EF-hand type of Ca2+ binding site was under-represented in the data set of structures analysed as far as its biological distribution is concerned, but was adequately represented for the chemical survey undertaken. A turn or loop structure was found to provide the bulk of the ligands to Ca2+, but helix and sheet secondary structures are slightly better providers of bidentate carboxylate ligation than turn or loop structures. The average coordination number for Ca2+ was 6.0, though for EF-hand sites it is 7. The average coordination number of a lanthanide ion in an intrinsic protein Ca2+ site was 7.2, but for the adventitious sites was only 4.4. A survey of the Cambridge Structural Database showed there are small-molecule lanthanide complexes with low coordination numbers but it is likely that water molecules, which do not appear in the electron density maps, are present for some lanthanide sites in proteins. A detailed comparison of the well-defined Ca2+ and lanthanide ion binding sites suggests that a reduction of hydrogen bonding associated with the ligating residues of the binding sites containing lanthanide ions may be a response to the additional positive charge of the lanthanide ion. Major structural differences between Ca2+ binding sites with weak and strong binding affinities were not obvious, a consequence of long-range electrostatic interactions and metal ion-induced protein conformational changes modulating affinities.     

Dissecting molecular details and functional effects of the high‐affinity copper binding site in plasminogen activator Inhibitor‐1

Plasminogen activator inhibitor‐1 (PAI‐1) is the primary inhibitor for plasminogen activators, tissue‐type plasminogen activator (tPA) and urokinase‐type plasminogen activator (uPA). As a unique member in the serine protease inhibitor (serpin) family, PAI‐1 is metastable and converts to an inactive, latent structure with a half‐life of 1–2 hr under physiological conditions. Unusual effects of metals on the rate of the latency conversion are incompletely understood. Previous work has identified two residues near the N‐terminus, H2 and H3, which reside in a high‐affinity copper‐binding site in PAI‐1 [Bucci JC, McClintock CS, Chu Y, Ware GL, McConnell KD, Emerson JP, Peterson CB (2017) J Biol Inorg Chem 22:1123–1,135]. In this study, neighboring residues, H10, E81, and H364, were tested as possible sites that participate in Cu(II) coordination at the high‐affinity site. Kinetic methods, gel sensitivity assays, and isothermal titration calorimetry (ITC) revealed that E81 and H364 have different roles in coordinating metal and mediating the stability of PAI‐1. H364 provides a third histidine in the metal‐coordination sphere with H2 and H3. In contrast, E81 does not appear to be required for metal ligation along with histidines; contacts made by the side‐chain carboxylate upon metal binding are perturbed and, in turn, influence dynamic fluctuations within the region encompassing helices D, E, and F and the W86 loop that are important in the pathway for the PAI‐1 latency conversion. This investigation underscores a prominent role of protein dynamics, noncovalent bonding networks and ligand binding in controlling the stability of the active form of PAI‐1.     

Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19F NMR

R67 dihydrofolate reductase (R67 DHFR) is a plasmid‐encoded enzyme that confers resistance to the antibacterial drug trimethoprim. R67 DHFR is a tetramer with a single active site that is unusual as both cofactor and substrate are recognized by symmetry‐related residues. Such promiscuity has limited our previous efforts to differentiate ligand binding by NMR. To address this problem, we incorporated fluorine at positions 4, 5, 6, or 7 of the indole rings of tryptophans 38 and 45 and characterized the spectra to determine which probe was optimal for studying ligand binding. Two resonances were observed for all apo proteins. Unexpectedly, the W45 resonance appeared broad, and truncation of the disordered N‐termini resulted in the appearance of one sharp W45 resonance. These results are consistent with interaction of the N‐terminus with W45. Binding of the cofactor broadened W38 for all fluorine probes, whereas substrate, dihydrofolate, binding resulted in the appearance of three new resonances for 4‐ and 5‐fluoroindole labeled protein and severe line broadening for 6‐ and 7‐fluoroindole R67 DHFR. W45 became slightly broader upon ligand binding. With only two peaks in the ¹⁹F NMR spectra, our data were able to differentiate cofactor and substrate binding to the single, symmetric active site of R67 DHFR and yield binding affinities.     

Solution structures of the Shewanella woodyi H‐NOX protein in the presence and absence of soluble guanylyl cyclase stimulator IWP‐051

Heme‐nitric oxide/oxygen binding (H‐NOX) domains bind gaseous ligands for signal transduction in organisms spanning prokaryotic and eukaryotic kingdoms. In the bioluminescent marine bacterium Shewanella woodyi (Sw), H‐NOX proteins regulate quorum sensing and biofilm formation. In higher animals, soluble guanylyl cyclase (sGC) binds nitric oxide with an H‐NOX domain to induce cyclase activity and regulate vascular tone, wound healing and memory formation. sGC also binds stimulator compounds targeting cardiovascular disease. The molecular details of stimulator binding to sGC remain obscure but involve a binding pocket near an interface between H‐NOX and coiled‐coil domains. Here, we report the full NMR structure for CO‐ligated Sw H‐NOX in the presence and absence of stimulator compound IWP‐051, and its backbone dynamics. Nonplanar heme geometry was retained using a semi‐empirical quantum potential energy approach. Although IWP‐051 binding is weak, a single binding conformation was found at the interface of the two H‐NOX subdomains, near but not overlapping with sites identified in sGC. Binding leads to rotation of the subdomains and closure of the binding pocket. Backbone dynamics are similar across both domains except for two helix‐connecting loops, which display increased dynamics that are further enhanced by compound binding. Structure‐based sequence analyses indicate high sequence diversity in the binding pocket, but the pocket itself appears conserved among H‐NOX proteins. The largest dynamical loop lies at the interface between Sw H‐NOX and its binding partner as well as in the interface with the coiled coil in sGC, suggesting a critical role for the loop in signal transduction.     

Benchmarking a computational design method for the incorporation of metal ion‐binding sites at symmetric protein interfaces

The design of novel metal‐ion binding sites along symmetric axes in protein oligomers could provide new avenues for metalloenzyme design, construction of protein‐based nanomaterials and novel ion transport systems. Here, we describe a computational design method, symmetric protein recursive ion‐cofactor sampling (SyPRIS), for locating constellations of backbone positions within oligomeric protein structures that are capable of supporting desired symmetrically coordinated metal ion(s) chelated by sidechains (chelant model). Using SyPRIS on a curated benchmark set of protein structures with symmetric metal binding sites, we found high recovery of native metal coordinating rotamers: in 65 of the 67 (97.0%) cases, native rotamers featured in the best scoring model while in the remaining cases native rotamers were found within the top three scoring models. In a second test, chelant models were crossmatched against protein structures with identical cyclic symmetry. In addition to recovering all native placements, 10.4% (8939/86013) of the non‐native placements, had acceptable geometric compatibility scores. Discrimination between native and non‐native metal site placements was further enhanced upon constrained energy minimization using the Rosetta energy function. Upon sequence design of the surrounding first‐shell residues, we found further stabilization of native placements and a small but significant (1.7%) number of non‐native placement‐based sites with favorable Rosetta energies, indicating their designability in existing protein interfaces. The generality of the SyPRIS approach allows design of novel symmetric metal sites including with non‐natural amino acid sidechains, and should enable the predictive incorporation of a variety of metal‐containing cofactors at symmetric protein interfaces.     

Transition metal cation inhibition of Mycobacterium tuberculosis esterase RV0045C

Mycobacterium tuberculosis virulence is highly metal‐dependent with metal availability modulating the shift from the dormant to active states of M. tuberculosis infection. Rv0045c from M. tuberculosis is a proposed metabolic serine hydrolase whose folded stability is dependent on divalent metal concentration. Herein, we measured the divalent metal inhibition profile of the enzymatic activity of Rv0045c and found specific divalent transition metal cations (Cu²⁺ ≥ Zn²⁺ > Ni²⁺ > Co²⁺) strongly inhibited its enzymatic activity. The metal cations bind allosterically, largely affecting values for k _cat rather than K _M. Removal of the artificial N‐terminal 6xHis‐tag did not change the metal‐dependent inhibition, indicating that the allosteric inhibition site is native to Rv0045c. To isolate the site of this allosteric regulation in Rv0045c, the structures of Rv0045c were determined at 1.8 Å and 2.0 Å resolution in the presence and absence of Zn²⁺ with each structure containing a previously unresolved dynamic loop spanning the binding pocket. Through the combination of structural analysis with and without zinc and targeted mutagenesis, this metal‐dependent inhibition was traced to multiple chelating residues (H202A/E204A) on a flexible loop, suggesting dynamic allosteric regulation of Rv0045c by divalent metals. Although serine hydrolases like Rv0045c are a large and diverse enzyme superfamily, this is the first structural confirmation of allosteric regulation of their enzymatic activity by divalent metals.     

A useful epitope tag derived from maltose binding protein

Maltose binding protein (MBP) is used in recombinant protein expression as an affinity and solubility tag. The monoclonal antibody B48 binds MBP tightly and has no cross‐reactivity to other proteins in an Escherichia coli lysate. This high level of specificity suggested that MBP contains an epitope that could prove useful as a purification and visualization tag for proteins expressed in E. coli. To discover the MBP epitope, a co‐crystal structure was determined for MBP bound to its antibody and four amino acids of MBP were identified as critical for the binding interaction. Fusions of various fragments of MBP to the glutathione S‐transferase protein were engineered in order to identify the smallest fragment still recognized by the α‐MBP antibody. Stabilization of the epitope via mutational engineering resulted in a minimized 14 amino‐acid tag.     

High‐resolution analysis of the conformational transition of pro‐apoptotic Bak at the lipid membrane

Permeabilization of the outer mitochondrial membrane by pore‐forming Bcl2 proteins is a crucial step for the induction of apoptosis. Despite a large set of data suggesting global conformational changes within pro‐apoptotic Bak during pore formation, high‐resolution structural details in a membrane environment remain sparse. Here, we used NMR and HDX‐MS (Hydrogen deuterium exchange mass spectrometry) in lipid nanodiscs to gain important high‐resolution structural insights into the conformational changes of Bak at the membrane that are dependent on a direct activation by BH3‐only proteins. Furthermore, we determined the first high‐resolution structure of the Bak transmembrane helix. Upon activation, α‐helix 1 in the soluble domain of Bak dissociates from the protein and adopts an unfolded and dynamic potentially membrane‐bound state. In line with this finding, comparative protein folding experiments with Bak and anti‐apoptotic BclxL suggest that α‐helix 1 in Bak is a metastable structural element contributing to its pro‐apoptotic features. Consequently, mutagenesis experiments aimed at stabilizing α‐helix 1 yielded Bak variants with delayed pore‐forming activity. These insights will contribute to a better mechanistic understanding of Bak‐mediated membrane permeabilization.     

Accurate Prediction of Peptide Binding Sites on Protein Surfaces

Many important protein–protein interactions are mediated by the binding of a short peptide stretch in one protein to a large globular segment in another. Recent efforts have provided hundreds of examples of new peptides binding to proteins for which a three-dimensional structure is available (either known experimentally or readily modeled) but where no structure of the protein–peptide complex is known. To address this gap, we present an approach that can accurately predict peptide binding sites on protein surfaces. For peptides known to bind a particular protein, the method predicts binding sites with great accuracy, and the specificity of the approach means that it can also be used to predict whether or not a putative or predicted peptide partner will bind. We used known protein–peptide complexes to derive preferences, in the form of spatial position specific scoring matrices, which describe the binding-site environment in globular proteins for each type of amino acid in bound peptides. We then scan the surface of a putative binding protein for sites for each of the amino acids present in a peptide partner and search for combinations of high-scoring amino acid sites that satisfy constraints deduced from the peptide sequence. The method performed well in a benchmark and largely agreed with experimental data mapping binding sites for several recently discovered interactions mediated by peptides, including RG-rich proteins with SMN domains, Epstein-Barr virus LMP1 with TRADD domains, DBC1 with Sir2, and the Ago hook with Argonaute PIWI domain. The method, and associated statistics, is an excellent tool for predicting and studying binding sites for newly discovered peptides mediating critical events in biology.     

Enzyme catalysis prior to aromatic residues: Reverse engineering of a dephospho‐CoA kinase

The wide variety of protein structures and functions results from the diverse properties of the 20 canonical amino acids. The generally accepted hypothesis is that early protein evolution was associated with enrichment of a primordial alphabet, thereby enabling increased protein catalytic efficiencies and functional diversification. Aromatic amino acids were likely among the last additions to genetic code. The main objective of this study was to test whether enzyme catalysis can occur without the aromatic residues (aromatics) by studying the structure and function of dephospho‐CoA kinase (DPCK) following aromatic residue depletion. We designed two variants of a putative DPCK from Aquifex aeolicus by substituting (a) Tyr, Phe and Trp or (b) all aromatics (including His). Their structural characterization indicates that substituting the aromatics does not markedly alter their secondary structures but does significantly loosen their side chain packing and increase their sizes. Both variants still possess ATPase activity, although with 150–300 times lower efficiency in comparison with the wild‐type phosphotransferase activity. The transfer of the phosphate group to the dephospho‐CoA substrate becomes heavily uncoupled and only the His‐containing variant is still able to perform the phosphotransferase reaction. These data support the hypothesis that proteins in the early stages of life could support catalytic activities, albeit with low efficiencies. An observed significant contraction upon ligand binding is likely important for appropriate organization of the active site. Formation of firm hydrophobic cores, which enable the assembly of stably structured active sites, is suggested to provide a selective advantage for adding the aromatic residues.     

ScanMoment: a web server for combinatorial analysis of basic residues in nucleic acid binding sites

ScanMoment is a webserver designed to identify the presence of the basic faced α‐helix (BFAH) motif in the nucleic acid binding sites of proteins. The program calculates the ’Basic Moment‘, a parameter that quantitizes the distribution of basic residues on the surface of an α‐helix. A sliding window is used to generate a plot displaying regions of the protein sequence that possesses a high Basic Moment and hus likely to possess a BFAH motif. The user may vary the periodicity from that of an alpha‐helix (100°), to those of other secondary structures such as beta sheets and 3₁₀ helices. The program can also plot the periodicity of basic residues in a protein sequence using a Fourier transformation. The procedure has been used to characterize the presence of BFAHs in the N‐terminal extensions of the eukaryotic aminoacyl‐tRNA synthetases and to indicate the presence of a BFAH in the tRNA binding site of alanyl‐tRNA synthetase.     

Systematic discovery of linear binding motifs targeting an ancient protein interaction surface on MAP kinases

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.     

A Pareto-Optimal Refinement Method for Protein Design Scaffolds

Computational design of protein function involves a search for amino acids with the lowest energy subject to a set of constraints specifying function. In many cases a set of natural protein backbone structures, or “scaffolds”, are searched to find regions where functional sites (an enzyme active site, ligand binding pocket, protein – protein interaction region, etc.) can be placed, and the identities of the surrounding amino acids are optimized to satisfy functional constraints. Input native protein structures almost invariably have regions that score very poorly with the design force field, and any design based on these unmodified structures may result in mutations away from the native sequence solely as a result of the energetic strain. Because the input structure is already a stable protein, it is desirable to keep the total number of mutations to a minimum and to avoid mutations resulting from poorly-scoring input structures. Here we describe a protocol using cycles of minimization with combined backbone/sidechain restraints that is Pareto-optimal with respect to RMSD to the native structure and energetic strain reduction. The protocol should be broadly useful in the preparation of scaffold libraries for functional site design.     

Unveiling the binding mode of perfluorooctanoic acid to human serum albumin

Perfluorooctanoic acid (PFOA) is a toxic compound that is absorbed and distributed throughout the body by noncovalent binding to serum proteins such as human serum albumin (hSA). Though the interaction between PFOA and hSA has been already assessed using various analytical techniques, a high resolution and detailed analysis of the binding mode is still lacking. We report here the crystal structure of hSA in complex with PFOA and a medium‐chain saturated fatty acid (FA). A total of eight distinct binding sites, four occupied by PFOAs and four by FAs, have been identified. In solution binding studies confirmed the 4:1 PFOA‐hSA stoichiometry and revealed the presence of one high and three low affinity binding sites. Competition experiments with known hSA‐binding drugs allowed locating the high affinity binding site in sub‐domain IIIA. The elucidation of the molecular basis of the interaction between PFOA and hSA might provide not only a better assessment of the absorption and elimination mechanisms of these compounds in vivo but also have implications for the development of novel molecular receptors for diagnostic and biotechnological applications.     

Identification of lectin receptors for conserved SARS‐CoV‐2 glycosylation sites

New SARS‐CoV‐2 variants are continuously emerging with critical implications for therapies or vaccinations. The 22 N‐glycan sites of Spike remain highly conserved among SARS‐CoV‐2 variants, opening an avenue for robust therapeutic intervention. Here we used a comprehensive library of mammalian carbohydrate‐binding proteins (lectins) to probe critical sugar residues on the full‐length trimeric Spike and the receptor binding domain (RBD) of SARS‐CoV‐2. Two lectins, Clec4g and CD209c, were identified to strongly bind to Spike. Clec4g and CD209c binding to Spike was dissected and visualized in real time and at single‐molecule resolution using atomic force microscopy. 3D modelling showed that both lectins can bind to a glycan within the RBD‐ACE2 interface and thus interferes with Spike binding to cell surfaces. Importantly, Clec4g and CD209c significantly reduced SARS‐CoV‐2 infections. These data report the first extensive map and 3D structural modelling of lectin‐Spike interactions and uncovers candidate receptors involved in Spike binding and SARS‐CoV‐2 infections. The capacity of CLEC4G and mCD209c lectins to block SARS‐CoV‐2 viral entry holds promise for pan‐variant therapeutic interventions.     

NMR hawk‐eyed view of AlphaFold2 structures

The prediction of the three‐dimensional (3D) structure of proteins from the amino acid sequence made a stunning breakthrough reaching atomic accuracy. Using the neural network‐based method AlphaFold2, 3D structures of almost the entire human proteome have been predicted and made available (https://www.alphafold.ebi.ac.uk). To gain insight into how well AlphaFold2 structures represent the conformation of proteins in solution, I here compare the AlphaFold2 structures of selected small proteins with their 3D structures that were determined by nuclear magnetic resonance (NMR) spectroscopy. Proteins were selected for which the 3D solution structures were determined on the basis of a very large number of distance restraints and residual dipolar couplings and are thus some of the best‐resolved solution structures of proteins to date. The quality of the backbone conformation of the AlphaFold2 structures is assessed by fitting a large set of experimental residual dipolar couplings (RDCs). The analysis shows that experimental RDCs fit extremely well to the AlphaFold2 structures predicted for GB3, DinI, and ubiquitin. In the case of GB3, the accuracy of the AlphaFold2 structure even surpasses that of a 1.1 Å crystal structure. Fitting of experimental RDCs furthermore allows identification of AlphaFold2 structures that are best representative of the protein''s conformation in solution as seen for the EF hands of the N‐terminal domain of Ca²⁺‐ligated calmodulin. Taken together, the analysis shows that structures predicted by AlphaFold2 can be highly representative of the solution conformation of proteins. The combination of AlphaFold2 structures with RDCs promises to be a powerful approach to study structural changes in proteins.     

A unified statistical potential reveals that amino acid stickiness governs nonspecific recruitment of client proteins into condensates

Membraneless organelles are cellular compartments that form by liquid–liquid phase separation of one or more components. Other molecules, such as proteins and nucleic acids, will distribute between the cytoplasm and the liquid compartment in accordance with the thermodynamic drive to lower the free energy of the system. The resulting distribution colocalizes molecular species to carry out a diversity of functions. Two factors could drive this partitioning: the difference in solvation between the dilute versus dense phase and intermolecular interactions between the client and scaffold proteins. Here, we develop a set of knowledge‐based potentials that allow for the direct comparison between stickiness, which is dominated by desolvation energy, and pairwise residue contact propensity terms. We use these scales to examine experimental data from two systems: protein cargo dissolving within phase‐separated droplets made from FG repeat proteins of the nuclear pore complex and client proteins dissolving within phase‐separated FUS droplets. These analyses reveal a close agreement between the stickiness of the client proteins and the experimentally determined values of the partition coefficients (R > 0.9), while pairwise residue contact propensities between client and scaffold show weaker correlations. Hence, the stickiness of client proteins is sufficient to explain their differential partitioning within these two phase‐separated systems without taking into account the composition of the condensate. This result implies that selective trafficking of client proteins to distinct membraneless organelles requires recognition elements beyond the client sequence composition.StatementEmpirical potentials for amino acid stickiness and pairwise residue contact propensities are derived. These scales are unique in that they enable direct comparison of desolvation versus contact terms. We find that partitioning of a client protein to a condensate is best explained by amino acid stickiness.