Assembly of the Tn7 targeting complex by a regulated stepwise process.

The Tn7 family of transposons is notable for its highly regulated integration mechanisms, including programmable RNA-guided transposition. The targeting pathways rely on dedicated target selection proteins from the TniQ family and the AAA+ adaptor TnsC to recruit and activate the transposase at specific target sites. Here, we report the cryoelectron microscopy (cryo-EM) structures of TnsC bound to the TniQ domain of TnsD from prototypical Tn7 and unveil key regulatory steps stemming from unique behaviors of ATP- versus ADP-bound TnsC. We show that TnsD recruits ADP-bound dimers of TnsC and acts as an exchange factor to release one protomer with exchange to ATP. This loading process explains how TnsC assembles a heptameric ring unidirectionally from the target site. This unique loading process results in functionally distinct TnsC protomers within the ring, providing a checkpoint for target immunity and explaining how insertions at programmed sites precisely occur in a specific orientation across Tn7 elements.

Evolutionary conservation of the structure and function of meiotic Rec114-Mei4 and Mer2 complexes.

Meiosis-specific Rec114-Mei4 and Mer2 complexes are thought to enable Spo11-mediated DNA double-strand break (DSB) formation through a mechanism that involves DNA-dependent condensation. However, the structure, molecular properties, and evolutionary conservation of Rec114-Mei4 and Mer2 are unclear. Here, we present AlphaFold models of Rec114-Mei4 and Mer2 complexes supported by nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), and mutagenesis. We show that dimers composed of the Rec114 C terminus form α-helical chains that cup an N-terminal Mei4 α helix, and that Mer2 forms a parallel homotetrameric coiled coil. Both Rec114-Mei4 and Mer2 bind preferentially to branched DNA substrates, indicative of multivalent protein-DNA interactions. Indeed, the Rec114-Mei4 interaction domain contains two DNA-binding sites that point in opposite directions and drive condensation. The Mer2 coiled-coil domain bridges coaligned DNA duplexes, likely through extensive electrostatic interactions along the length of the coiled coil. Finally, we show that the structures of Rec114-Mei4 and Mer2 are conserved across eukaryotes, while DNA-binding properties vary significantly. This work provides insights into the mechanism whereby Rec114-Mei4 and Mer2 complexes promote the assembly of the meiotic DSB machinery and suggests a model in which Mer2 condensation is the essential driver of assembly, with the DNA-binding activity of Rec114-Mei4 playing a supportive role.

Bispecific Forkhead Transcription Factor FoxN3 Recognizes Two Distinct Motifs with Different DNA Shapes.

Transcription factors (TFs) control gene expression by binding DNA recognition sites in genomic regulatory regions. Although most forkhead TFs recognize a canonical forkhead (FKH) motif, RYAAAYA, some forkheads recognize a completely different (FHL) motif, GACGC. Bispecific forkhead proteins recognize both motifs, but the molecular basis for bispecific DNA recognition is not understood. We present co-crystal structures of the FoxN3 DNA binding domain bound to the FKH and FHL sites, respectively. FoxN3 adopts a similar conformation to recognize both motifs, making contacts with different DNA bases using the same amino acids. However, the DNA structure is different in the two complexes. These structures reveal how a single TF binds two unrelated DNA sequences and the importance of DNA shape in the mechanism of bispecific recognition.

Structures of Bacterial RNA Polymerase Complexes Reveal the Mechanism of DNA Loading and Transcription Initiation.

Gene transcription is carried out by multi-subunit RNA polymerases (RNAPs). Transcription initiation is a dynamic multi-step process that involves the opening of the double-stranded DNA to form a transcription bubble and delivery of the template strand deep into the RNAP for RNA synthesis. Applying cryoelectron microscopy to a unique transcription system using σ54 (σN), the major bacterial variant sigma factor, we capture a new intermediate state at 4.1 Å where promoter DNA is caught at the entrance of the RNAP cleft. Combining with new structures of the open promoter complex and an initial de novo transcribing complex at 3.4 and 3.7 Å, respectively, our studies reveal the dynamics of DNA loading and mechanism of transcription bubble stabilization that involves coordinated, large-scale conformational changes of the universally conserved features within RNAP and DNA. In addition, our studies reveal a novel mechanism of strand separation by σ54.

A molecular mechanism for the "digital" response of p53 to stress.

The tumor suppressor protein p53 accumulates in response to cellular stress and consequently orchestrates the expression of multiple genes in a p53-level and time-dependent manner to overcome stress consequences, for which a molecular mechanism is currently unknown. Previously, we reported that DNA torsional flexibility distinguishes among p53 response elements (REs) and that transactivation at basal p53 levels is correlated with p53 REs flexibility. Here, we calculated the flexibility of ~200 p53 REs. By connecting functional outcomes of p53-target genes' activation to the calculated flexibility of their REs, we show that genes known to belong to pathways that are activated rapidly upon stress contain REs that are significantly more flexible relative to REs of genes known to be involved in pathways that are activated later in the response to stress. The global structural properties of several p53 REs belonging to different pathways were experimentally validated. Additionally, reporter-gene expression driven by flexible p53 REs occurred at lower p53 levels and with faster rates than expression from rigid REs. Furthermore, analysis of published endogenous mRNA levels of p53-target genes as a function of REs' flexibility showed that early versus late genes differ significantly in their flexibility properties of their REs and that highly flexible p53 REs enable high-activation level exclusively to early-response genes. Overall, we demonstrate that DNA flexibility of p53 REs contributes significantly to functional selectivity in the p53 system by facilitating the initial steps of p53-dependent target-genes expression, thereby contributing to survival versus death decisions in the p53 system.

The cellular Notch1 protein promotes KSHV reactivation in an Rta-dependent manner.

The cellular Notch signal transduction pathway is intimately associated with infections by Kaposi's sarcoma-associated herpesvirus (KSHV) and other gamma-herpesviruses. RBP-Jk, the cellular DNA binding component of the canonical Notch pathway, is the key Notch downstream effector protein in virus-infected and uninfected animal cells. Reactivation of KSHV from latency requires the viral lytic switch protein, Rta, to form complexes with RBP-Jk on numerous sites within the viral DNA. Constitutive Notch activity is essential for KSHV pathophysiology in models of Kaposi's sarcoma (KS) and Primary Effusion Lymphoma (PEL), and we demonstrate that Notch1 is also constitutively active in infected Vero cells. Although the KSHV genome contains >100 RBP-Jk DNA motifs, we show that none of the four isoforms of activated Notch can productively reactivate the virus from latency in a highly quantitative trans-complementing reporter virus system. Nevertheless, Notch contributed positively to reactivation because broad inhibition of Notch1-4 with gamma-secretase inhibitor (GSI) or expression of dominant negative mastermind-like1 (dnMAML1) coactivators severely reduced production of infectious KSHV from Vero cells. Reduction of KSHV production is associated with gene-specific reduction of viral transcription in both Vero and PEL cells. Specific inhibition of Notch1 by siRNA partially reduces the production of infectious KSHV, and NICD1 forms promoter-specific complexes with viral DNA during reactivation. We conclude that constitutive Notch activity is required for the robust production of infectious KSHV, and our results implicate activated Notch1 as a pro-viral member of a MAML1/RBP-Jk/DNA complex during viral reactivation. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) manipulates the host cell oncogenic Notch signaling pathway for viral reactivation from latency and cell pathogenesis. KSHV reactivation requires that the viral protein Rta functionally interacts with RBP-Jk, the DNA-binding component of the Notch pathway, and with promoter DNA to drive transcription of productive cycle genes. We show that the Notch pathway is constitutively active during KSHV reactivation and is essential for robust production of infectious virus progeny. Inhibiting Notch during reactivation reduces the expression of specific viral genes yet does not affect the growth of the host cells. Although Notch cannot reactivate KSHV alone, the requisite expression of Rta reveals a previously unappreciated role for Notch in reactivation. We propose that activated Notch cooperates with Rta in a promoter-specific manner that is partially programmed by Rta's ability to redistribute RBP-Jk DNA binding to the virus during reactivation.

Studies of Local DNA Backbone Conformation and Conformational Disorder Using Site-Specific Exciton-Coupled Dimer Probe Spectroscopy.

The processes of genome expression, regulation, and repair require direct interactions between proteins and DNA at specific sites located at and near single-stranded-double-stranded DNA (ssDNA-dsDNA) junctions. Here, we review the application of recently developed spectroscopic methods and analyses that combine linear absorbance and circular dichroism spectroscopy with nonlinear 2D fluorescence spectroscopy to study the local conformations and conformational disorder of the sugar-phosphate backbones of ssDNA-dsDNA fork constructs that have been internally labeled with exciton-coupled cyanine (iCy3)2 dimer probes. With the application of these methods, the (iCy3)2 dimer can serve as a reliable probe of the mean local conformations and conformational distributions of the sugar-phosphate backbones of dsDNA at various critical positions. The results of our studies suggest a possible structural framework for understanding the roles of DNA breathing in driving the processes of protein-DNA complex assembly and function.

Studying Macromolecular Interactions of Cellular Machines by the Combined Use of Analytical Ultracentrifugation, Light Scattering, and Fluorescence Spectroscopy Methods.

Cellular machines formed by the interaction and assembly of macromolecules are essential in many processes of the living cell. These assemblies involve homo- and hetero-associations, including protein-protein, protein-DNA, protein-RNA, and protein-polysaccharide associations, most of which are reversible. This chapter describes the use of analytical ultracentrifugation, light scattering, and fluorescence-based methods, well-established biophysical techniques, to characterize interactions leading to the formation of macromolecular complexes and their modulation in response to specific or unspecific factors. We also illustrate, with several examples taken from studies on bacterial processes, the advantages of the combined use of subsets of these techniques as orthogonal analytical methods to analyze protein oligomerization and polymerization, interactions with ligands, hetero-associations involving membrane proteins, and protein-nucleic acid complexes.

Variable Inhibition of DNA Unwinding Rates Catalyzed by the SARS-CoV-2 Helicase Nsp13 by Structurally Distinct Single DNA Lesions.

The SARS-CoV-2 helicase, non-structural protein 13 (Nsp13), plays an essential role in viral replication, translocating in the 5' → 3' direction as it unwinds double-stranded RNA/DNA. We investigated the impact of structurally distinct DNA lesions on DNA unwinding catalyzed by Nsp13. The selected lesions include two benzo[a]pyrene (B[a]P)-derived dG adducts, the UV-induced cyclobutane pyrimidine dimer (CPD), and the pyrimidine (6-4) pyrimidone (6-4PP) photolesion. The experimentally observed unwinding rate constants (kobs) and processivities (P) were examined. Relative to undamaged DNA, the kobs values were diminished by factors of up to ~15 for B[a]P adducts but only by factors of ~2-5 for photolesions. A minor-groove-oriented B[a]P adduct showed the smallest impact on P, which decreased by ~11% compared to unmodified DNA, while an intercalated one reduced P by ~67%. However, the photolesions showed a greater impact on the processivities; notably, the CPD, with the highest kobs value, exhibited the lowest P, which was reduced by ~90%. Our findings thus show that DNA unwinding efficiencies are lesion-dependent and most strongly inhibited by the CPD, leading to the conclusion that processivity is a better measure of DNA lesions' inhibitory effects than unwinding rate constants.

Recognition of extracellular DNA by type IV pili promotes biofilm formation by Clostridioides difficile.

Clostridioides difficile is a Gram-positive bacillus, which is a frequent cause of gastrointestinal infections triggered by the depletion of the gut microbiome. Because of the frequent recurrence of these infections after antibiotic treatment, mechanisms of C. difficile persistence and recurrence, including biofilm formation, are of increasing interest. Previously, our group and others found that type IV pili, filamentous helical appendages polymerized from protein subunits, promoted microcolony and biofilm formation in C. difficile. In Gram-negative bacteria, the ability of type IV pili to mediate bacterial self-association has been explained through interactions between the pili of adjacent cells, but type IV pili from several Gram-negative species are also required for natural competence through DNA uptake. Here, we report the ability of two C. difficile pilin subunits, PilJ and PilW, to bind to DNA in vitro, as well as the defects in biofilm formation in the pilJ and pilW gene-interruption mutants. Additionally, we have resolved the X-ray crystal structure of PilW, which we use to model possible structural mechanisms for the formation of C. difficile biofilm through interactions between type IV pili and the DNA of the extracellular matrix. Taken together, our results provide further insight into the relationship between type IV pilus function and biofilm formation in C. difficile and, more broadly, suggest that DNA recognition by type IV pili and related structures may have functional importance beyond DNA uptake for natural competence.

The Chromatin Regulator HMGA1a Undergoes Phase Separation in the Nucleus.

The protein high mobility group A1 (HMGA1) is an important regulator of chromatin organization and function. However, the mechanisms by which it exerts its biological function are not fully understood. Here, we report that the HMGA isoform, HMGA1a, nucleates into foci that display liquid-like properties in the nucleus, and that the protein readily undergoes phase separation to form liquid condensates in vitro. By bringing together machine-leaning modelling, cellular and biophysical experiments and multiscale simulations, we demonstrate that phase separation of HMGA1a is promoted by protein-DNA interactions, and has the potential to be modulated by post-transcriptional effects such as phosphorylation. We further show that the intrinsically disordered C-terminal tail of HMGA1a significantly contributes to its phase separation through electrostatic interactions via AT hooks 2 and 3. Our work sheds light on HMGA1 phase separation as an emergent biophysical factor in regulating chromatin structure.

DNAgenie: accurate prediction of DNA-type-specific binding residues in protein sequences.

Efforts to elucidate protein-DNA interactions at the molecular level rely in part on accurate predictions of DNA-binding residues in protein sequences. While there are over a dozen computational predictors of the DNA-binding residues, they are DNA-type agnostic and significantly cross-predict residues that interact with other ligands as DNA binding. We leverage a custom-designed machine learning architecture to introduce DNAgenie, first-of-its-kind predictor of residues that interact with A-DNA, B-DNA and single-stranded DNA. DNAgenie uses a comprehensive physiochemical profile extracted from an input protein sequence and implements a two-step refinement process to provide accurate predictions and to minimize the cross-predictions. Comparative tests on an independent test dataset demonstrate that DNAgenie outperforms the current methods that we adapt to predict residue-level interactions with the three DNA types. Further analysis finds that the use of the second (refinement) step leads to a substantial reduction in the cross predictions. Empirical tests show that DNAgenie's outputs that are converted to coarse-grained protein-level predictions compare favorably against recent tools that predict which DNA-binding proteins interact with double-stranded versus single-stranded DNAs. Moreover, predictions from the sequences of the whole human proteome reveal that the results produced by DNAgenie substantially overlap with the known DNA-binding proteins while also including promising leads for several hundred previously unknown putative DNA binders. These results suggest that DNAgenie is a valuable tool for the sequence-based characterization of protein functions. The DNAgenie's webserver is available at http://biomine.cs.vcu.edu/servers/DNAgenie/.

Unusual Relationship between Iron Deprivation and Organophosphate Hydrolase Expression.

Organophosphate hydrolases (OPH), hitherto known to hydrolyze the third ester bond of organophosphate (OP) insecticides and nerve agents, have recently been shown to interact with outer membrane transport components, namely, TonB and ExbB/ExbD. In an OPH negative background, Sphingopyxis wildii cells failed to transport ferric enterobactin and showed retarded growth under iron-limiting conditions. We now show the OPH-encoding organophosphate degradation (opd) gene from Sphingobium fuliginis ATCC 27551 to be part of the iron regulon. A fur-box motif found to be overlapping with the transcription start site (TSS) of the opd gene coordinates with an iron responsive element (IRE) RNA motif identified in the 5' coding region of the opd mRNA to tightly regulate opd gene expression. The fur-box motif serves as a target for the Fur repressor in the presence of iron. A decrease in iron concentration leads to the derepression of opd. IRE RNA inhibits the translation of opd mRNA and serves as a target for apo-aconitase (IRP). The IRP recruited by the IRE RNA abrogates IRE-mediated translational inhibition. Our findings establish a novel, multilayered, iron-responsive regulation that is crucial for OPH function in the transport of siderophore-mediated iron uptake. IMPORTANCE Sphingobium fuliginis, a soil-dwelling microbe isolated from agricultural soils, was shown to degrade a variety of insecticides and pesticides. These synthetic chemicals function as potent neurotoxins, and they belong to a class of chemicals termed organophosphates. S. fuliginis codes for OPH, an enzyme that has been shown to be involved in the metabolism of several organophosphates and their derivatives. Interestingly, OPH has also been shown to facilitate siderophore-mediated iron uptake in S. fuliginis and in another Sphingomonad, namely, Sphingopyxis wildii, implying that this organophosphate-metabolizing protein has a role in iron homeostasis, as well. Our research dissects the underlying molecular mechanisms linking iron to the expression of OPH, prompting a reconsideration of the role of OPH in Sphingomonads and a reevaluation of the evolutionary origins of the OPH proteins from soil bacteria.

Basic residues at the C-gate of DNA gyrase are involved in DNA supercoiling.

DNA gyrase is a type II topoisomerase that is responsible for maintaining the topological state of bacterial and some archaeal genomes. It uses an ATP-dependent two-gate strand-passage mechanism that is shared among all type II topoisomerases. During this process, DNA gyrase creates a transient break in the DNA, the G-segment, to form a cleavage complex. This allows a second DNA duplex, known as the T-segment, to pass through the broken G-segment. After the broken strand is religated, the T-segment is able to exit out of the enzyme through a gate called the C-gate. Although many steps of the type II topoisomerase mechanism have been studied extensively, many questions remain about how the T-segment ultimately exits out of the C-gate. A recent cryo-EM structure of Streptococcus pneumoniae GyrA shows a putative T-segment in close proximity to the C-gate, suggesting that residues in this region may be important for coordinating DNA exit from the enzyme. Here, we show through site-directed mutagenesis and biochemical characterization that three conserved basic residues in the C-gate of DNA gyrase are important for DNA supercoiling activity, but not for ATPase or cleavage activity. Together with the structural information previously published, our data suggest a model in which these residues cluster to form a positively charged region that facilitates T-segment passage into the cavity formed between the DNA gate and C-gate.

Analysis of the DNA-binding properties of Alx1, an evolutionarily conserved regulator of skeletogenesis in echinoderms.

Alx1, a homeodomain-containing transcription factor, is a highly conserved regulator of skeletogenesis in echinoderms. In sea urchins, Alx1 plays a central role in the differentiation of embryonic primary mesenchyme cells (PMCs) and positively regulates the transcription of most biomineralization genes expressed by these cells. The alx1 gene arose via duplication and acquired a skeletogenic function distinct from its paralog (alx4) through the exonization of a 41-amino acid motif (the D2 domain). Alx1 and Alx4 contain glutamine-50 paired-type homeodomains, which interact preferentially with palindromic binding sites in vitro. Chromatin immunoprecipitation sequencing (ChIP-seq) studies have shown, however, that Alx1 binds both to palindromic and half sites in vivo. To address this apparent discrepancy and explore the function of the D2 domain, we used an endogenous cis-regulatory module associated with Sp-mtmmpb, a gene that encodes a PMC-specific metalloprotease, to analyze the DNA-binding properties of Alx1. We find that Alx1 forms dimeric complexes on TAAT-containing half sites by a mechanism distinct from the well-known mechanism of dimerization on palindromic sites. We used transgenic reporter assays to analyze the functional roles of half sites in vivo and demonstrate that two sites with partially redundant functions are essential for the PMC-specific activity of the Sp-mtmmpb cis-regulatory module. Finally, we show that the D2 domain influences the DNA-binding properties of Alx1 in vitro, suggesting that the exonization of this motif may have facilitated the acquisition of new transcriptional targets and consequently a novel developmental function.

Archaeal DNA replication.

DNA replication is essential for all life forms. Although the process is fundamentally conserved in the three domains of life, bioinformatic, biochemical, structural, and genetic studies have demonstrated that the process and the proteins involved in archaeal DNA replication are more similar to those in eukaryal DNA replication than in bacterial DNA replication, but have some archaeal-specific features. The archaeal replication system, however, is not monolithic, and there are some differences in the replication process between different species. In this review, the current knowledge of the mechanisms governing DNA replication in Archaea is summarized. The general features of the replication process as well as some of the differences are discussed.

Differential functions of FANCI and FANCD2 ubiquitination stabilize ID2 complex on DNA.

The Fanconi anaemia (FA) pathway is a dedicated pathway for the repair of DNA interstrand crosslinks and is additionally activated in response to other forms of replication stress. A key step in the FA pathway is the monoubiquitination of each of the two subunits (FANCI and FANCD2) of the ID2 complex on specific lysine residues. However, the molecular function of these modifications has been unknown for nearly two decades. Here, we find that ubiquitination of FANCD2 acts to increase ID2's affinity for double-stranded DNA via promoting a large-scale conformational change in the complex. The resulting complex encircles DNA, by forming a secondary "Arm" ID2 interface. Ubiquitination of FANCI, on the other hand, largely protects the ubiquitin on FANCD2 from USP1-UAF1 deubiquitination, with key hydrophobic residues of FANCI's ubiquitin being important for this protection. In effect, both of these post-translational modifications function to stabilize a conformation in which the ID2 complex encircles DNA.

Cognate DNA Recognition by Engrailed Homeodomain Involves a Conformational Change Controlled via an Electrostatic-Spring-Loaded Latch.

Transcription factors must scan genomic DNA, recognize the cognate sequence of their control element(s), and bind tightly to them. The DNA recognition process is primarily carried out by their DNA binding domains (DBD), which interact with the cognate site with high affinity and more weakly with any other DNA sequence. DBDs are generally thought to bind to their cognate DNA without changing conformation (lock-and-key). Here, we used nuclear magnetic resonance and circular dichroism to investigate the interplay between DNA recognition and DBD conformation in the engrailed homeodomain (enHD), as a model case for the homeodomain family of eukaryotic DBDs. We found that the conformational ensemble of enHD is rather flexible and becomes gradually more disordered as ionic strength decreases following a Debye-Hückel's dependence. Our analysis indicates that enHD's response to ionic strength is mediated by a built-in electrostatic spring-loaded latch that operates as a conformational transducer. We also found that, at moderate ionic strengths, enHD changes conformation upon binding to cognate DNA. This change is of larger amplitude and somewhat orthogonal to the response to ionic strength. As a consequence, very high ionic strengths (e.g., 700 mM) block the electrostatic-spring-loaded latch and binding to cognate DNA becomes lock-and-key. However, the interplay between enHD conformation and cognate DNA binding is robust across a range of ionic strengths (i.e., 45 to 300 mM) that covers the physiologically-relevant conditions. Therefore, our results demonstrate the presence of a mechanism for the conformational control of cognate DNA recognition on a eukaryotic DBD. This mechanism can function as a signal transducer that locks the DBD in place upon encountering the cognate site during active DNA scanning. The electrostatic-spring-loaded latch of enHD can also enable the fine control of DNA recognition in response to transient changes in local ionic strength induced by variate physiological processes.

Alkoxylalkyl Esters of Nucleotide Analogs Inhibit Polyomavirus DNA Replication and Large T Antigen Activities.

Polyomavirus infections occur commonly in humans and are normally nonfatal. However, in immunocompromised individuals, they are intractable and frequently fatal. Due to a lack of approved drugs to treat polyomavirus infections, cidofovir, a phosphonate nucleotide analog approved to treat cytomegalovirus infections, has been repurposed as an antipolyomavirus agent. Cidofovir has been modified in various ways to improve its efficacies as a broad-spectrum antiviral agent. However, the actual mechanisms and targets of cidofovir and its modified derivatives as antipolyomavirus agents are still under research. Here, polyomavirus large tumor antigen (Tag) activities were identified as the viral target of cidofovir derivatives. The alkoxyalkyl ester derivatives of cidofovir efficiently inhibit polyomavirus DNA replication in cell-free human extracts and a viral in vitro replication system utilizing only purified proteins. We present evidence that DNA helicase and DNA binding activities of polyomavirus Tags are diminished in the presence of low concentrations of alkoxyalkyl ester derivatives of cidofovir, suggesting that the inhibition of viral DNA replication is at least in part mediated by inhibiting single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) binding activities of Tags. These findings show that the alkoxyalkyl ester derivatives of cidofovir are effective in vitro without undergoing further conversions, and we conclude that the inhibitory mechanisms of nucleotide analog-based drugs are more complex than previously believed.

Comprehensive review and empirical analysis of hallmarks of DNA-, RNA- and protein-binding residues in protein chains.

Proteins interact with a variety of molecules including proteins and nucleic acids. We review a comprehensive collection of over 50 studies that analyze and/or predict these interactions. While majority of these studies address either solely protein-DNA or protein-RNA binding, only a few have a wider scope that covers both protein-protein and protein-nucleic acid binding. Our analysis reveals that binding residues are typically characterized with three hallmarks: relative solvent accessibility (RSA), evolutionary conservation and propensity of amino acids (AAs) for binding. Motivated by drawbacks of the prior studies, we perform a large-scale analysis to quantify and contrast the three hallmarks for residues that bind DNA-, RNA-, protein- and (for the first time) multi-ligand-binding residues that interact with DNA and proteins, and with RNA and proteins. Results generated on a well-annotated data set of over 23 000 proteins show that conservation of binding residues is higher for nucleic acid- than protein-binding residues. Multi-ligand-binding residues are more conserved and have higher RSA than single-ligand-binding residues. We empirically show that each hallmark discriminates between binding and nonbinding residues, even predicted RSA, and that combining them improves discriminatory power for each of the five types of interactions. Linear scoring functions that combine these hallmarks offer good predictive performance of residue-level propensity for binding and provide intuitive interpretation of predictions. Better understanding of these residue-level interactions will facilitate development of methods that accurately predict binding in the exponentially growing databases of protein sequences.