Molecular characterization of Streptococcus suis isolates recovered from diseased pigs in Europe.

Streptococcus suis is a major swine pathogen and zoonotic agent, causing important economic losses to the porcine industry. Here, we used genomics approaches to characterize 251 S. suis isolates recovered from diseased pigs across Belgium, France, Germany, Hungary, the Netherlands, Spain, and the United Kingdom. We identified 13 serotypes, being serotypes 9 and 2 the most prevalent, and 34 sequence types (STs), including 16 novel STs, although ST16 and ST1 dominated the strain population. Phylogenetic analysis revealed complex genetic relationships, notable geographic clustering, and potential differential capacity for capsular switching among serotype 9 isolates. We found antimicrobial resistance (AMR) genes in 85.3% of the isolates, with high frequencies of genes conferring resistance to tetracyclines and macrolides. Specifically, 49.4% of the isolates harbored the tetO gene, and 64.9% possessed the ermB gene. Additionally, we observed a diverse array of virulence-associated genes (VAGs), including the classical VAGs mrp, epf, and sly, with variable presence across different genotypes. The high genetic diversity among European S. suis isolates highlights the importance of targeted antimicrobial use and flexible vaccine strategies. Rapid strain characterization is crucial for optimizing swine health management, enabling tailored interventions like the development of autovaccines to mitigate S. suis infections.

Distal Protein-Protein Interactions Contribute to SARS-CoV-2 Main Protease Substrate Binding and Nirmatrelvir Resistance.

SARS-CoV-2 main protease, M pro , is responsible for the processing of the viral polyproteins into individual proteins, including the protease itself. M pro is a key target of anti-COVID-19 therapeutics such as nirmatrelvir (the active component of Paxlovid). Resistance mutants identified clinically and in viral passage assays contain a combination of active site mutations (e.g. E166V, E166A, L167F), which reduce inhibitor binding and enzymatic activity, and non-active site mutations (e.g. P252L, T21I, L50F), which restore the fitness of viral replication. Although the mechanism of resistance for the active site mutations is apparent, the role of the non-active site mutations in fitness rescue remains elusive. In this study, we use the model system of a M pro triple mutant (L50F/E166A/L167F) that confers not only nirmatrelvir drug resistance but also a similar fitness of replication compared to the wild-type both in vitro and in vivo. By comparing peptide and full-length M pro protein as substrates, we demonstrate that the binding of M pro substrate involves more than residues in the active site. In particular, L50F and other non-active site mutations can enhance the M pro dimer-dimer interactions and help place the nsp5-6 substrate at the enzyme catalytic center. The structural and enzymatic activity data of M pro L50F, L50F/E166A/L167F, and others underscore the importance of considering the whole substrate protein in studying M pro and substrate interactions, and offers important insights into M pro function, resistance development, and inhibitor design.

Naturally Occurring Mutations of SARS-CoV-2 Main Protease Confer Drug Resistance to Nirmatrelvir.

The SARS-CoV-2 main protease (Mpro) is the drug target of Pfizer's oral drug nirmatrelvir. The emergence of SARS-CoV-2 variants with mutations in Mpro raised the alarm of potential drug resistance. To identify potential clinically relevant drug-resistant mutants, we systematically characterized 102 naturally occurring Mpro mutants located at 12 residues at the nirmatrelvir-binding site, among which 22 mutations in 5 residues, including S144M/F/A/G/Y, M165T, E166 V/G/A, H172Q/F, and Q192T/S/L/A/I/P/H/V/W/C/F, showed comparable enzymatic activity to the wild-type (kcat/Km < 10-fold change) while being resistant to nirmatrelvir (Ki > 10-fold increase). X-ray crystal structures were determined for six representative mutants with and/or without GC-376/nirmatrelvir. Using recombinant SARS-CoV-2 viruses generated from reverse genetics, we confirmed the drug resistance in the antiviral assay and showed that Mpro mutants with reduced enzymatic activity had attenuated viral replication. Overall, our study identified several drug-resistant hotspots in Mpro that warrant close monitoring for possible clinical evidence of nirmatrelvir resistance, some of which have already emerged in independent viral passage assays conducted by others.

A yeast-based system to study SARS-CoV-2 Mpro structure and to identify nirmatrelvir resistant mutations.

The SARS-CoV-2 main protease (Mpro) is a major therapeutic target. The Mpro inhibitor, nirmatrelvir, is the antiviral component of Paxlovid, an orally available treatment for COVID-19. As Mpro inhibitor use increases, drug resistant mutations will likely emerge. We have established a non-pathogenic system, in which yeast growth serves as an approximation for Mpro activity, enabling rapid identification of mutants with altered enzymatic activity and drug sensitivity. The E166 residue is known to be a potential hot spot for drug resistance and yeast assays identified substitutions which conferred strong nirmatrelvir resistance and others that compromised activity. On the other hand, N142A and the P132H mutation, carried by the Omicron variant, caused little to no change in drug response and activity. Standard enzymatic assays confirmed the yeast results. In turn, we solved the structures of Mpro E166R, and Mpro E166N, providing insights into how arginine may drive drug resistance while asparagine leads to reduced activity. The work presented here will help characterize novel resistant variants of Mpro that may arise as Mpro antivirals become more widely used.

Naturally occurring mutations of SARS-CoV-2 main protease confer drug resistance to nirmatrelvir.

The SARS-CoV-2 main protease (M pro ) is the drug target of Pfizer’s oral drug Paxlovid. The emergence of SARS-CoV-2 variants with mutations in M pro raised the alarm of potential drug resistance. In this study, we identified 100 naturally occurring M pro mutations located at the nirmatrelvir binding site, among which 20 mutants, including S144M/F/A/G/Y, M165T, E166G, H172Q/F, and Q192T/S/L/A/I/P/H/V/W/C/F, showed comparable enzymatic activity to the wild-type (k cat /K m <10-fold change) and resistance to nirmatrelvir (K i >10-fold increase). X-ray crystal structures were determined for seven representative mutants with and/or without GC-376/nirmatrelvir. Viral growth assay showed that M pro mutants with reduced enzymatic activity led to attenuated viral replication. Overall, our study identified several drug resistant hot spots that warrant close monitoring for possible clinical evidence of Paxlovid resistance. One Sentence Summary: Paxlovid resistant SARS-CoV-2 viruses with mutations in the main protease have been identified from clinical isolates.

A yeast-based system to study SARS-CoV-2 Mpro structure and to identify nirmatrelvir resistant mutations.

The SARS-CoV-2 main protease (Mpro) is a major therapeutic target. The Mpro inhibitor, nirmatrelvir, is the antiviral component of Paxlovid, an orally available treatment for COVID-19. As Mpro inhibitor use increases, drug resistant mutations will likely emerge. We have established a non-pathogenic system, in which yeast growth serves as a proxy for Mpro activity, enabling rapid identification of mutants with altered enzymatic activity and drug sensitivity. The E166 residue is known to be a potential hot spot for drug resistance and yeast assays showed that an E166R substitution conferred strong nirmatrelvir resistance while an E166N mutation compromised activity. On the other hand, N142A and P132H mutations caused little to no change in drug response and activity. Standard enzymatic assays confirmed the yeast results. In turn, we solved the structures of Mpro E166R, and Mpro E166N, providing insights into how arginine may drive drug resistance while asparagine leads to reduced activity. The work presented here will help characterize novel resistant variants of Mpro that may arise as Mpro antivirals become more widely used.

A yeast-based system to study SARS-CoV-2 M pro structure and to identify nirmatrelvir resistant mutations.

The SARS-CoV-2 main protease (M pro ) is a major therapeutic target. The M pro inhibitor, nirmatrelvir, is the antiviral component of Paxlovid, an orally available treatment for COVID-19. As M pro inhibitor use increases, drug resistant mutations will likely emerge. We have established a non-pathogenic system, in which yeast growth serves as a proxy for M pro activity, enabling rapid identification of mutants with altered enzymatic activity and drug sensitivity. The E166 residue is known to be a potential hot spot for drug resistance and yeast assays showed that an E166R substitution conferred strong nirmatrelvir resistance while an E166N mutation compromised activity. On the other hand, N142A and P132H mutations caused little to no change in drug response and activity. Standard enzymatic assays confirmed the yeast results. In turn, we solved the structures of M pro E166R, and M pro E166N, providing insights into how arginine may drive drug resistance while asparagine leads to reduced activity. The work presented here will help characterize novel resistant variants of M pro that may arise as M pro antivirals become more widely used.

A unique class of Zn2+-binding serine-based PBPs underlies cephalosporin resistance and sporogenesis in Clostridioides difficile.

Treatment with ß-lactam antibiotics, particularly cephalosporins, is a major risk factor for Clostridioides difficile infection. These broad-spectrum antibiotics irreversibly inhibit penicillin-binding proteins (PBPs), which are serine-based enzymes that assemble the bacterial cell wall. However, C. difficile has four different PBPs (PBP1-3 and SpoVD) with various roles in growth and spore formation, and their specific links to ß-lactam resistance in this pathogen are underexplored. Here, we show that PBP2 (known to be essential for vegetative growth) is the primary bactericidal target for ß-lactams in C. difficile. PBP2 is insensitive to cephalosporin inhibition, and this appears to be the main basis for cephalosporin resistance in this organism. We determine crystal structures of C. difficile PBP2, alone and in complex with ß-lactams, revealing unique features including ligand-induced conformational changes and an active site Zn2+-binding motif that influences ß-lactam binding and protein stability. The Zn2+-binding motif is also present in C. difficile PBP3 and SpoVD (which are known to be essential for sporulation), as well as in other bacterial taxa including species living in extreme environments and the human gut. We speculate that this thiol-containing motif and its cognate Zn2+ might function as a redox sensor to regulate cell wall synthesis for survival in adverse or anaerobic environments.

High-performance superconducting quantum processors via laser annealing of transmon qubits.

Scaling the number of qubits while maintaining high-fidelity quantum gates remains a key challenge for quantum computing. Presently, superconducting quantum processors with >50 qubits are actively available. For these systems, fixed-frequency transmons are attractive because of their long coherence and noise immunity. However, scaling fixed-frequency architectures proves challenging because of precise relative frequency requirements. Here, we use laser annealing to selectively tune transmon qubits into desired frequency patterns. Statistics over hundreds of annealed qubits demonstrate an empirical tuning precision of 18.5 MHz, with no measurable impact on qubit coherence. We quantify gate error statistics on a tuned 65-qubit processor, with median two-qubit gate fidelity of 98.7%. Baseline tuning statistics yield a frequency-equivalent resistance precision of 4.7 MHz, sufficient for high-yield scaling beyond 103 qubit levels. Moving forward, we anticipate selective laser annealing to play a central role in scaling fixed-frequency architectures.

Structural Insights into Molecular Recognition by Human Chemokine CCL19.

The human chemokines CCL19 and CCL21 bind to the G protein-coupled receptor (GPCR) CCR7 and play an important role in the trafficking of immune cells as well as cancer metastasis. Conserved binding sites for sulfotyrosine residues on the receptor contribute significantly to the chemokine/GPCR interaction and have been shown to provide promising targets for new drug-discovery efforts to disrupt the chemokine/GPCR interaction and, consequently, tumor metastasis. Here, we report the first X-ray crystal structure of a truncated CCL19 (residues 7-70) at 2.50 Å resolution, revealing molecular details crucial for protein-protein interactions. Although the overall structure is similar to the previously determined NMR model, there are important variations, particularly near the N terminus and the so-called 30's and 40's loops. Computational analysis using the FTMap server indicates the potential importance of these areas in ligand binding and the differences in binding hotspots compared to CCL21. NMR titration experiments using a CCR7-derived peptide (residues 5-11, TDDYIGD) further demonstrate potential receptor recognition sites, such as those near the C terminus and 40's loop, which consist of both positively charged and hydrophobic residues that may be important for receptor binding. Taken together, the X-ray, NMR, and computational analysis herein provide insights into the overall structure and molecular features of CCL19 and enables investigation into this chemokine's function and inhibitor development.

Development of a Benzothiazole Scaffold-Based Androgen Receptor N-Terminal Inhibitor for Treating Androgen-Responsive Prostate Cancer.

All current clinically approved androgen deprivation therapies for prostate cancer target the C-terminal ligand-binding domain of the androgen receptor (AR). However, the main transactivation function of the receptor is localized at the AR N-terminal domain (NTD). Targeting the AR NTD directly is a challenge because of its intrinsically disordered structure and the lack of pockets for drugs to bind. Here, we have taken an alternative approach using the cochaperone BAG1L, which interacts with the NTD, to develop a novel AR inhibitor. We describe the identification of 2-(4-fluorophenyl)-5-(trifluoromethyl)-1,3-benzothiazole (A4B17), a small molecule that inhibits BAG1L-AR NTD interaction, attenuates BAG1L-mediated AR NTD activity, downregulates AR target gene expression, and inhibits proliferation of AR-positive prostate cancer cells. This compound represents a prototype of AR antagonists that could be key in the development of future prostate cancer therapeutics.

Low barrier hydrogen bonds in protein structure and function.

Low-barrier hydrogen bonds (LBHBs) are a special type of short hydrogen bond (HB) that is characterized by the equal sharing of a hydrogen atom. The existence and catalytic role of LBHBs in proteins has been intensely contested. Advancements in X-ray and neutron diffraction methods has revealed delocalized hydrogen atoms involved in potential LBHBs in a number of proteins, while also demonstrating that short HBs are not necessarily LBHBs. More importantly, a series of experiments on ketosteroid isomerase (KSI) have suggested that LBHBs are significantly stronger than standard HBs in the protein microenvironment in terms of enthalpy, but not free energy. The discrepancy between the enthalpy and free energy of LBHBs offers clues to the challenges, and potential solutions, of the LBHB debate, where the unique strength of LBHBs plays a special role in the kinetic processes of enzyme function and structure, together with other molecular forces in a pre-organized environment.

Metallocenyl 7-ACA Conjugates: Antibacterial Activity Studies and Atomic-Resolution X-ray Crystal Structure with CTX-M ß-Lactamase.

The conjugation of organometallic groups to current ß-lactam antibiotics is a field of increasing study due to the ability of certain organometallic groups to enhance the antibiotic potency of these drugs. Herein, we report the antibacterial properties of two metallocenyl (ferrocenyl and ruthenocenyl) 7-aminocephalosporanic acid (7-ACA) antibiotic conjugates. Continuing a trend we found in our previous studies, the ruthenocenyl conjugate showed greater antibacterial activity than its ferrocenyl counterpart. Compared with the previously published 7-aminodesacetoxycephalosporanic acid (7-ADCA) conjugates, the 3-acetyloxymethyl group significantly improved the compounds' activity. Furthermore, the Rc-7-ACA compound was more active against clinical Staphylococcus aureus isolates than the ampicillin reference. Noticeably, neither of the two new compounds showed an undesirable toxic effect in HeLa and L929 cells at the concentrations at which they displayed strong antibacterial effects. The antibacterial activity of the two metallocenyl 7-ACA derivatives was further confirmed by scanning electron microscopy (SEM). SEM micrographs showed that bacteria treated with metallocenyl 7-ACA derivatives feature cell wall damage and morphology changes. Using a CTX-M-14 ß-lactamase competition assay based on nitrocefin hydrolysis, we showed that the Rc-7-ACA bound more favorably to CTX-M-14 than its ferrocenyl counterpart, again confirming the superiority of the ruthenocenyl moiety over the ferrocenyl one in interacting with proteins. We also report a 1.47 Å resolution crystal structure of Rc-7-ACA in complex with the CTX-M-14 E166A mutant, an enzyme sharing a similar active site configuration with penicillin-binding proteins, the molecular target of ß-lactam antibiotics. These results strengthen the case for the antibacterial utility of the Rc and Fc groups.

Characterization of Arylalkylamine N-Acyltransferase from Tribolium castaneum: An Investigation into a Potential Next-Generation Insecticide Target.

The growing issue of insecticide resistance has meant the identification of novel insecticide targets has never been more important. Arylalkylamine N-acyltransferases (AANATs) have been suggested as a potential new target. These promiscuous enzymes are involved in the N-acylation of biogenic amines to form N-acylamides. In insects, this process is a key step in melanism, hardening of the cuticle, removal of biogenic amines, and in the biosynthesis of fatty acid amides. The unique nature of each AANAT isoform characterized indicates each organism accommodates an assembly of discrete AANATs relatively exclusive to that organism. This implies a high potential for selectivity in insecticide design, while also maintaining polypharmacology. Presented here is a thorough kinetic and structural analysis of AANAT found in one of the most common secondary pests of all plant commodities in the world, Tribolium castaneum. The enzyme, named TcAANAT0, catalyzes the formation of short-chain N-acylarylalkylamines, with short-chain acyl-CoAs (C2-C10), benzoyl-CoA, and succinyl-CoA functioning in the role of acyl donor. Recombinant TcAANAT0 was expressed and purified from E. coli and was used to investigate the kinetic and chemical mechanism of catalysis. The kinetic mechanism is an ordered sequential mechanism with the acyl-CoA binding first. pH-rate profiles and site-directed mutagenesis studies identified amino acids critical to catalysis, providing insights about the chemical mechanism of TcAANAT0. A crystal structure was obtained for TcAANAT0 bound to acetyl-CoA, revealing valuable information about its active site. This combination of kinetic analysis and crystallography alongside mutagenesis and sequence analysis shines light on some approaches possible for targeting TcAANAT0 and other AANATs for novel insecticide design.

Differential long-term regulation of TAS2R14 by structurally distinct agonists.

Bitter taste receptor-14 (TAS2R14) is a GPCR also expressed on human airway smooth muscle cells, which signals to intracellular [Ca2+], resulting in relaxation of the airway, and is a novel target for bronchodilators. Here, we examine long-term, agonist-promoted down-regulation of TAS2R14 expression because tachyphylaxis would be an undesirable therapeutic characteristic. Five TAS2R structurally distinct full agonists were studied to ascertain biasing away from down-regulation. Agonist exposure for 18 h caused minimal desensitization by diphenhydramine (DPD) compared with ∼50% desensitization with all other agonists. Agonists evoked ß-arrestin recruitment to TAS2R14, which was not seen with a phosphoacceptor-deficient mutant, TAS2R14-10A. All agonists except for DPD also caused subsequent TAS2R14 internalization and trafficking via early and late endosomes to down-regulation. TAS2R14-10A failed to undergo these events with any agonist. Molecular docking showed that DPD has specific interactions deep within a binding pocket that are not observed with the other agonists, which may lock the receptor in a conformation that does not internalize and therefore does not undergo down-regulation. Thus, TAS2R14 is subject to ß-arrestin-mediated internalization and subsequent down-regulation with chronic exposure to most agonists. However, by manipulating the agonist structure, biasing toward G-protein coupling but away from long-term down-regulation can be achieved.-Woo, J. A., Castaño, M., Goss, A., Kim, D., Lewandowski, E. M., Chen, Y., Liggett, S. B. Differential long-term regulation of TAS2R14 by structurally distinct agonists.

Mechanisms of proton relay and product release by Class A ß-lactamase at ultrahigh resolution.

The ß-lactam antibiotics inhibit penicillin-binding proteins (PBPs) by forming a stable, covalent, acyl-enzyme complex. During the evolution from PBPs to Class A ß-lactamases, the ß-lactamases acquired Glu166 to activate a catalytic water and cleave the acyl-enzyme bond. Here we present three product complex crystal structures of CTX-M-14 Class A ß-lactamase with a ruthenocene-conjugated penicillin-a 0.85 Å resolution structure of E166A mutant complexed with the penilloate product, a 1.30 Å resolution complex structure of the same mutant with the penicilloate product, and a 1.18 Å resolution complex structure of S70G mutant with a penicilloate product epimer-shedding light on the catalytic mechanisms and product inhibition of PBPs and Class A ß-lactamases. The E166A-penilloate complex captured the hydrogen bonding network following the protonation of the leaving group and, for the first time, unambiguously show that the ring nitrogen donates a proton to Ser130, which in turn donates a proton to Lys73. These observations indicate that in the absence of Glu166, the equivalent lysine would be neutral in PBPs and therefore capable of serving as the general base to activate the catalytic serine. Together with previous results, this structure suggests a common proton relay network shared by Class A ß-lactamases and PBPs, from the catalytic serine to the lysine, and ultimately to the ring nitrogen. Additionally, the E166A-penicilloate complex reveals previously unseen conformational changes of key catalytic residues during the release of the product, and is the first structure to capture the hydrolyzed product in the presence of an unmutated catalytic serine. DATABASE: Structural data are available in the PDB database under the accession numbers 5TOP, 5TOY, and 5VLE.

Structural and Mechanistic Analysis of Drosophila melanogaster Agmatine N-Acetyltransferase, an Enzyme that Catalyzes the Formation of N-Acetylagmatine.

Agmatine N-acetyltransferase (AgmNAT) catalyzes the formation of N-acetylagmatine from acetyl-CoA and agmatine. Herein, we provide evidence that Drosophila melanogaster AgmNAT (CG15766) catalyzes the formation of N-acetylagmatine using an ordered sequential mechanism; acetyl-CoA binds prior to agmatine to generate an AgmNAT•acetyl-CoA•agmatine ternary complex prior to catalysis. Additionally, we solved a crystal structure for the apo form of AgmNAT with an atomic resolution of 2.3 Å, which points towards specific amino acids that may function in catalysis or active site formation. Using the crystal structure, primary sequence alignment, pH-activity profiles, and site-directed mutagenesis, we evaluated a series of active site amino acids in order to assign their functional roles in AgmNAT. More specifically, pH-activity profiles identified at least one catalytically important, ionizable group with an apparent pKa of ~7.5, which corresponds to the general base in catalysis, Glu-34. Moreover, these data led to a proposed chemical mechanism, which is consistent with the structure and our biochemical analysis of AgmNAT.

Antibacterial Properties of Metallocenyl-7-ADCA Derivatives and Structure in Complex with CTX-M ß-Lactamase.

A series of six novel metallocenyl-7-ADCA (metallocenyl = ferrocenyl or ruthenocenyl; 7-ADCA = 7-aminodesacetoxycephalosporanic acid) conjugates were synthesized and their antibacterial properties evaluated by biochemical and microbiological assays. The ruthenocene derivatives showed a higher level of inhibition of DD-carboxypeptidase 64-575, a Penicillin Binding Protein (PBP), than the ferrocene derivatives and the reference compound penicillin G. Protein X-ray crystallographic analysis revealed a covalent acyl-enzyme complex of a ruthenocenyl compound with CTX-M ß-lactamase E166A mutant, corresponding to a similar complex with PBPs responsible for the bactericidal activities of these compounds. Most interestingly, an intact compound was captured at the crystal-packing interface, elucidating for the first time the structure of a metallocenyl ß-lactam compound that previously eluded small molecule crystallography. We propose that protein crystals, even from biologically unrelated molecules, can be utilized to determine structures of small molecules.

Combined Molecular Dynamics Simulation-Molecular-Thermodynamic Theory Framework for Predicting Surface Tensions.

A molecular modeling approach is presented with a focus on quantitative predictions of the surface tension of aqueous surfactant solutions. The approach combines classical Molecular Dynamics (MD) simulations with a molecular-thermodynamic theory (MTT) [ Y. J. Nikas, S. Puvvada, D. Blankschtein, Langmuir 1992 , 8 , 2680 ]. The MD component is used to calculate thermodynamic and molecular parameters that are needed in the MTT model to determine the surface tension isotherm. The MD/MTT approach provides the important link between the surfactant bulk concentration, the experimental control parameter, and the surfactant surface concentration, the MD control parameter. We demonstrate the capability of the MD/MTT modeling approach on nonionic alkyl polyethylene glycol surfactants at the air-water interface and observe reasonable agreement of the predicted surface tensions and the experimental surface tension data over a wide range of surfactant concentrations below the critical micelle concentration. Our modeling approach can be extended to ionic surfactants and their mixtures with both ionic and nonionic surfactants at liquid-liquid interfaces.

Crystallographic Structure of Truncated CCL21 and the Putative Sulfotyrosine-Binding Site.

CCL21 chemokine binds the G protein-coupled receptor CCR7, aiding not only in immune response but also in cancer metastasis. Compared with other chemokines, CCL21 has a unique extended unstructured C-terminus that is truncated in some naturally occurring variants. We have determined the X-ray crystallographic structure of a truncated CCL21 (residues 1-79) lacking the extended C-terminus and identified, via two-dimensional nuclear magnetic resonance (NMR), a putative sulfotyrosine-binding site that may recognize such post-translationally modified tyrosine residues on the receptor. Compared to the previously determined NMR structure of full-length CCL21, the crystal structure presents new druggable binding hot spots resulting from an alternative N-loop conformation. In addition, whereas the previous NMR structure did not provide any structural information after residue 70, the C-terminus of the truncated CCL21, ordered up to Ala77 in our crystal structure, is placed near the N-loop and sulfotyrosine-binding site, indicating that the extended C-terminus of full-length CCL21 can interact with this important region for receptor binding. These observations suggest a potential origin for the autoinhibition of CCL21 activity that was recently described. The new crystal structure and binding hot spot analysis have important implications for the function of the CCL21 C-terminus and drug discovery.