RrA, an enzyme from Rhodospirillum rubrum, is a prototype of a new family of short-chain L-asparaginases.

L-Asparaginases (ASNases) catalyze the hydrolysis of L-Asn to L-Asp and ammonia. Members of the ASNase family are used as drugs in the treatment of leukemia, as well as in the food industry. The protomers of bacterial ASNases typically contain 300-400 amino acids (typical class 1 ASNases). In contrast, the chain of ASNase from Rhodospirillum rubrum, reported here and referred to as RrA, consists of only 172 amino acid residues. RrA is homologous to the N-terminal domain of typical bacterial class 1 ASNases and exhibits millimolar affinity for L-Asn. In this study, we demonstrate that RrA belongs to a unique family of cytoplasmic, short-chain ASNases (scASNases). These proteins occupy a distinct region in the sequence space, separate from the regions typically assigned to class 1 ASNases. The scASNases are present in approximately 7% of eubacterial species, spanning diverse bacterial lineages. They seem to be significantly enriched in species that encode for more than one class 1 ASNase. Here, we report biochemical, biophysical, and structural properties of RrA, a member of scASNases family. Crystal structures of the wild-type RrA, both with and without bound L-Asp, as well as structures of several RrA mutants, reveal topologically unique tetramers. Moreover, the active site of one protomer is complemented by two residues (Tyr21 and Asn26) from another protomer. Upon closer inspection, these findings clearly outline scASNases as a stand-alone subfamily of ASNases that can catalyze the hydrolysis of L-Asn to L-Asp despite the lack of the C-terminal domain that is present in all ASNases described structurally to date.

Mechanism of selective recognition of Lys48-linked polyubiquitin by macrocyclic peptide inhibitors of proteasomal degradation.

Post-translational modification of proteins with polyubiquitin chains is a critical cellular signaling mechanism in eukaryotes with implications in various cellular states and processes. Unregulated ubiquitin-mediated protein degradation can be detrimental to cellular homeostasis, causing numerous diseases including cancers. Recently, macrocyclic peptides were developed that selectively target long Lysine-48-linked polyubiquitin chains (tetra-ubiquitin) to inhibit ubiquitin-proteasome system, leading to attenuation of tumor growth in vivo. However, structural determinants of the chain length and linkage selectivity by these cyclic peptides remained unclear. Here, we uncover the mechanism underlying cyclic peptide's affinity and binding selectivity by combining X-ray crystallography, solution NMR, and biochemical studies. We found that the peptide engages three consecutive ubiquitins that form a ring around the peptide and determined requirements for preferential selection of a specific trimer moiety in longer polyubiquitin chains. The structural insights gained from this work will guide the development of next-generation cyclic peptides with enhanced anti-cancer activity.

The dimeric form of bacterial l-asparaginase YpAI is fully active.

l-asparaginases from mesophilic bacteria (ASNases), including two enzymes very successfully used in the treatment of leukaemia, have been consistently described as homotetramers. On the contrary, structural studies show that homodimers of these enzymes should be sufficient to carry out the catalytic reaction. In this report, we investigated whether the type I Yersinia pestis asparaginase (YpAI) is active in a dimeric form or whether the tetrameric quaternary structure is critical for its activity. Using multiple biophysical techniques that investigate enzymatic properties and quaternary structure at either high or low protein concentration, we found that dimeric YpAI is fully active, suggesting that the tetrameric form of this subfamily of enzymes does not bear significant enzymatic relevance. In this process, we extensively characterized YpAI, showing that it is a cooperative enzyme, although the mechanism of allostery is still not definitely established. We showed that, like most type I ASNases, the substrate affinity of YpAI is low and this enzyme is very similar in terms of both the structure and enzymatic properties to homologous type I ASNase from Escherichia coli (EcAI). We extended these studies to more medically relevant type II ASNases, used as anti-leukaemia drugs. We confirmed that type II ASNases are not allosteric, and that they might also be functional in a dimeric form. However, the determination of the accurate tetramer⇆dimer dissociation constants of these enzymes that most likely lie in the picomolar range is not possible with currently available biophysical techniques.

The E. coli L-asparaginase V27T mutant: structural and functional characterization and comparison with theoretical predictions.

Bacterial L-asparaginases have been used for over 40 years as anticancer drugs. Ardalan et al. (Medical Hypotheses 112, 7-17, 2018) proposed that the V27T mutant of Escherichia coli type II L-asparaginase, EcAII(V27T), should display altered biophysical and catalytic properties compared to the wild-type enzyme, EcAII(wt), rendering it more favourable as a pharmaceutical. They postulated that EcAII(V27T) would exhibit reduced glutaminolytic activity and be more stable compared to EcAII(wt). Their postulates, however, were purely theoretical. Here, we characterized experimentally selected properties of EcAII(V27T). We found asparaginolytic activity of this mutant unchanged, whereas its glutaminolytic activity was fourfold lower compared with EcAII(wt). We did not observe significant differences in stabilities of EcAII(wt) and EcAII(V27T). Crystal structures of the complexes with L-Asp and L-Glu showed considerable differences in binding modes of both substrates.

Structural and biochemical properties of L-asparaginase.

l-Asparaginase (a hydrolase converting l-asparagine to l-aspartic acid) was the first enzyme to be used in clinical practice as an anticancer agent after its approval in 1978 as a component of a treatment protocol for childhood acute lymphoblastic leukemia. Structural and biochemical properties of l-asparaginases have been extensively investigated during the last half-century, providing an accurate structural description of the enzyme isolated from a variety of sources, as well as clarifying the mechanism of its activity. This review provides a critical assessment of the current state of knowledge of primarily structural, but also selected biochemical properties of 'bacterial-type' l-asparaginases from different organisms. The most extensively studied members of this enzyme family are l-asparaginases highly homologous to one of the two enzymes from Escherichia coli (usually referred to as EcAI and EcAII). Members of this enzyme family, although often called bacterial-type l-asparaginases, have been also identified in such divergent organisms as archaea or eukarya. Over 100 structural models of l-asparaginases have been deposited in the Protein Data Bank during the last 30 years. One of the prime achievements of structure-centered approaches was the elucidation of the details of the mechanism of enzymatic action of this unique hydrolase that utilizes a side chain of threonine as the primary nucleophile. The molecular basis of other important properties of these enzymes, such as their substrate specificity, is still being evaluated. Results of structural and mechanistic studies of l-asparaginases are being utilized in efforts to improve the clinical properties of this important anticancer drug.

Generalized enzymatic mechanism of catalysis by tetrameric L-asparaginases from mesophilic bacteria.

The mechanism of catalysis by the L-glutaminase-asparaginase from Pseudomonas 7A (PGA) was investigated using structural, mass spectrometry, and kinetic data. We had previously proposed mechanism of hydrolysis of L-Asn by the type II L-asparaginase from E. coli (EcAII), but that work was limited to just one enzyme. Based on results presented in this report, we postulate that all homotetrameric L-asparaginases from mesophilic bacteria utilize a common ping-pong mechanism of catalysis consisting of two subsequent nucleophilic substitutions. Several new structures of non-covalent complexes of PGA with different substrates, as well as structures of covalent acyl-enzyme intermediates of PGA with canonical substrates (L-Asp and L-Glu) and an opportunistic ligand, a citrate anion, were determined. The results of kinetic experiments monitored by high-resolution LC/MS, when combined with new structural data, clearly show that the reaction catalyzed by L-glutaminase-asparaginases proceeds through formation of a covalent intermediate, as observed previously for EcAII. Additionally, by showing that the same mechanism applies to L-Asn and L-Gln, we postulate that it is common for all these structurally related enzymes.

Mechanism of Catalysis by l-Asparaginase.

Two bacterial type II l-asparaginases, from Escherichia coli and Dickeya chrysanthemi, have played a critical role for more than 40 years as therapeutic agents against juvenile leukemias and lymphomas. Despite a long history of successful pharmacological applications and the apparent simplicity of the catalytic reaction, controversies still exist regarding major steps of the mechanism. In this report, we provide a detailed description of the reaction catalyzed by E. coli type II l-asparaginase (EcAII). Our model was developed on the basis of new structural and biochemical experiments combined with previously published data. The proposed mechanism is supported by quantum chemistry calculations based on density functional theory. We provide strong evidence that EcAII catalyzes the reaction according to the double-displacement (ping-pong) mechanism, with formation of a covalent intermediate. Several steps of catalysis by EcAII are unique when compared to reactions catalyzed by other known hydrolytic enzymes. Here, the reaction is initiated by a weak nucleophile, threonine, without direct assistance of a general base, although a distant general base is identified. Furthermore, tetrahedral intermediates formed during the catalytic process are stabilized by a never previously described motif. Although the scheme of the catalytic mechanism was developed only on the basis of data obtained from EcAII and its variants, this novel mechanism of enzymatic hydrolysis could potentially apply to most (and possibly all) l-asparaginases.

Opportunistic complexes of E. coli L-asparaginases with citrate anions.

Active sites of enzymes are highly optimized for interactions with specific substrates, thus binding of opportunistic ligands is usually observed only in the absence of native substrates or products. However, during growth of crystals required for structure determination enzymes are often exposed to conditions significantly divergent from the native ones, leading to binding of unexpected ligands to active sites even in the presence of substrates. Failing to recognize this possibility may lead to incorrect interpretation of experimental results and to faulty conclusions. Here, we present several examples of binding of a citrate anion to the active sites of E. coli L-asparaginases I and II, even in the presence of the native substrate, L-Asn. A part of this report focuses on a comprehensive re-interpretation of structural results published previously for complexes of type I L-asparaginase (EcAI) from E. coli. In two re-refined structures a citrate anion forms an acyl-enzyme reaction intermediate with the catalytic threonine. These results emphasize the importance of careful and critical analysis during interpretation of crystallographic data.

Geometric considerations support the double-displacement catalytic mechanism of l-asparaginase.

Twenty crystal structures of the complexes of l-asparaginase with l-Asn, l-Asp, and succinic acid that are currently available in the Protein Data Bank, as well as 11 additional structures determined in the course of this project, were analyzed in order to establish the level of conservation of the geometric parameters describing interactions between the substrates and the active site of the enzymes. We found that such stereochemical relationships are highly conserved, regardless of the organism from which the enzyme was isolated, specific crystallization conditions, or the nature of the ligands. Analysis of the geometry of the interactions, including Bürgi-Dunitz and Flippin-Lodge angles, indicated that Thr12 (Escherichia coli asparaginase II numbering) is optimally placed to be the primary nucleophile in the most likely scenario utilizing a double-displacement mechanism, whereas catalysis through a single-displacement mechanism appears to be the least likely.

Crystal Structure of the Labile Complex of IL-24 with the Extracellular Domains of IL-22R1 and IL-20R2.

Crystal structure of the ternary complex of human IL-24 with two receptors, IL-22R1 and IL-20R2, has been determined at 2.15 Å resolution. A crystallizable complex was created by a novel approach involving fusing the ligand with a flexible linker to the presumed low-affinity receptor, and coexpression of this construct in Drosophila S2 cells together with the presumed high-affinity receptor. This approach, which may be generally applicable to other multiprotein complexes with low-affinity components, was necessitated by the instability of IL-24 expressed by itself in either bacteria or insect cells. Although IL-24 expressed in Escherichia coli was unstable and precipitated almost immediately upon its refolding and purification, a small fraction of IL-24 remaining in the folded state was shown to be active in a cell-based assay. In the crystal structure presented here, we found that two cysteine residues in IL-24 do not form a predicted disulfide bond. Lack of structural restraint by disulfides, present in other related cytokines, is most likely reason for the low stability of IL-24. Although the contact area between IL-24 and IL-22R1 is larger than between the cytokine and IL-20R2, calculations show the latter interaction to be slightly more stable, suggesting that the shared receptor (IL-20R2) might be the higher-affinity receptor.

Structural analysis and unique molecular recognition properties of a Bauhinia forficata lectin that inhibits cancer cell growth.

Lectins have been used at length for basic research and clinical applications. New insights into the molecular recognition properties enhance our basic understanding of carbohydrate-protein interactions and aid in the design/development of new lectins. In this study, we used a combination of cell-based assays, glycan microarrays, and X-ray crystallography to evaluate the structure and function of the recombinant Bauhinia forficata lectin (BfL). The lectin was shown to be cytostatic for several cancer cell lines included in the NCI-60 panel; in particular, it inhibited growth of melanoma cancer cells (LOX IMVI) by over 95%. BfL is dimeric in solution and highly specific for binding of oligosaccharides and glycopeptides with terminal N-acetylgalactosamine (GalNAc). BfL was found to have especially strong binding (apparent Kd  = 0.5-1.0 nm) to the tumor-associated Tn antigen. High-resolution crystal structures were determined for the ligand-free lectin, as well as for its complexes with three Tn glycopeptides, globotetraose, and the blood group A antigen. Extensive analysis of the eight crystal structures and comparison to structures of related lectins revealed several unique features of GalNAc recognition. Of special note, the carboxylate group of Glu126, lining the glycan-binding pocket, forms H-bonds with both the N-acetyl of GalNAc and the peptide amido group of Tn antigens. Stabilization provided by Glu126 is described here for the first time for any GalNAc-specific lectin. Taken together, the results provide new insights into the molecular recognition of carbohydrates and provide a structural understanding that will enable rational engineering of BfL for a variety of applications. DATABASE: Structural data are available in the PDB under the accession numbers 5T50, 5T52, 5T55, 5T54, 5T5L, 5T5J, 5T5P, and 5T5O.

Crystal Structure of a Complex of the Intracellular Domain of Interferon λ Receptor 1 (IFNLR1) and the FERM/SH2 Domains of Human JAK1.

The crystal structure of a construct consisting of the FERM and SH2-like domains of the human Janus kinase 1 (JAK1) bound to a fragment of the intracellular domain of the interferon-λ receptor 1 (IFNLR1) has been determined at the nominal resolution of 2.1Å. In this structure, the receptor peptide forms an 85-Å-long extended chain, in which both the previously identified box1 and box2 regions bind simultaneously to the FERM and SH2-like domains of JAK1. Both domains of JAK1 are generally well ordered, with regions not seen in the crystal structure limited to loops located away from the receptor-binding regions. The structure provides a much more complete and accurate picture of the interactions between JAK1 and IFNLR1 than those given in earlier reports, illuminating the molecular basis of the JAK-cytokine receptor association. A glutamate residue adjacent to the box2 region in IFNLR1 mimics the mode of binding of a phosphotyrosine in classical SH2 domains. It was shown here that a deletion of residues within the box1 region of the receptor abolishes stable interactions with JAK1, although it was previously shown that box2 alone is sufficient to stabilize a similar complex of the interferon-α receptor and TYK2.

Human ß-defensin 4 - defensin without the "twist".

ß-defensins are small, cysteine-rich, cationic peptides that contribute to various processes related to both arms of host defense, the innate and adaptive immunities. All ß-defensins are potent antimicrobials with activity targeting a broad range of pathogens. Some human ß-defensins (hBDs) are also capable of binding and activating specific chemokine receptors, leading to chemotaxis of receptor-presenting cells. Two receptors identified as targets of specific human ß-defensins are CCR2 and CCR6, both members of the seven-transmembrane family of chemokine receptors. Currently, around 50 open reading frames (ORFs) identified in the human genome encode proteins that have signatures characteristic of ß-defensins. Of those, only three, hBD1-3, have been thoroughly characterized to date, including a detailed structural description of their molecules. In addition, limited information on biological and bactericidal properties is available for hBD4, as well as the solution structure of hBD6. The crystal structure of hBD4, determined here at resolution of 1.60 Å, indicates significant structural differences between this molecule and those reported previously for other hBDs. Crystallographic studies indicate a possibility of unique dimerization of hBD4, confirmed by solution studies using analytical ultracentrifugation. In contrast to hBD1-3, hBD4 does not induce CCR6-mediated chemotaxis. This observation can be attributed to an unusual conformation of the hBD4 N-terminus. In agreement with previously published reports, hBD4 was shown to be a potent antibacterial agent, as demonstrated by results of assays with E. coli ATCC 25922 cells.

Design of composite inhibitors targeting glutamate carboxypeptidase II: the importance of effector functionalities.

Inhibitors targeting human glutamate carboxypeptidase II (GCPII) typically consist of a P1' glutamate-derived binding module, which warrants the high affinity and specificity, linked to an effector function that is positioned within the entrance funnel of the enzyme. Here we present a comprehensive structural and computational study aimed at dissecting the importance of the effector function for GCPII binding and affinity. To this end we determined crystal structures of human GCPII in complex with a series of phosphoramidate-based inhibitors harboring effector functions of diverse physicochemical characteristics. Our data show that higher binding affinities of phosphoramidates, compared to matching phosphonates, are linked to the presence of additional hydrogen bonds between Glu424 and Gly518 of the enzyme and the amide group of the phosphoramidate. While the positioning of the P1' glutamate-derived module within the S1' pocket of GCPII is invariant, interaction interfaces between effector functions and residues lining the entrance funnel are highly varied, with the positively charged arginine patch defined by Arg463, Arg534 and Arg536 being the only 'hot-spot' common to several studied complexes. This variability stems in part from the fact that the effector/GCPII interfaces generally encompass isolated areas of nonpolar residues within the entrance funnel and resulting van der Waals contacts lack the directionality typical for hydrogen bonding interactions. The presented data unravel a complexity of binding modes of inhibitors within non-prime site(s) of GCPII and can be exploited for the design of novel GCPII-specific compounds. PDB ID CODES: Atomic coordinates of the present structures together with the experimental structure factor amplitudes were deposited at the RCSB Protein Data Bank under accession codes 4P44 (complex with JRB-4-81), 4P45 (complex with JRB-4-73), 4P4B (complex with CTT54), 4P4D (complex with MP1C), 4P4E (complex with MP1D), 4P4F (complex with NC-2-40), 4P4I (complex with T33) and 4P4J (complex with T33D).

Structure of a lectin from the sea mussel Crenomytilus grayanus (CGL).

CGL is a 150 amino-acid residue lectin that was originally isolated from the sea mussel Crenomytilus grayanus. It is specific for binding GalNAc/Gal-containing carbohydrate moieties and in general does not share sequence homology with other known galectins or lectins. Since CGL displays antibacterial, antifungal and antiviral activities, and interacts with high affinity with mucin-type receptors, which are abundant on some cancer cells, knowledge of its structure is of significant interest. Conditions have been established for the expression, purification and crystallization of a recombinant variant of CGL. The crystal structure of recombinant CGL was determined and refined at a resolution of 2.12 Å. The amino-acid sequence of CGL contains three homologous regions (73% similarity) and the folded protein has a ß-trefoil topology. Structural comparison of CGL with the closely related lectin MytiLec allowed description of the glycan-binding pockets.

Structural and biochemical characterization of a novel aminopeptidase from human intestine.

N-acetylated α-linked acidic dipeptidase-like protein (NAALADase L), encoded by the NAALADL1 gene, is a close homolog of glutamate carboxypeptidase II, a metallopeptidase that has been intensively studied as a target for imaging and therapy of solid malignancies and neuropathologies. However, neither the physiological functions nor structural features of NAALADase L are known at present. Here, we report a thorough characterization of the protein product of the human NAALADL1 gene, including heterologous overexpression and purification, structural and biochemical characterization, and analysis of its expression profile. By solving the NAALADase L x-ray structure, we provide the first experimental evidence that it is a zinc-dependent metallopeptidase with a catalytic mechanism similar to that of glutamate carboxypeptidase II yet distinct substrate specificity. A proteome-based assay revealed that the NAALADL1 gene product possesses previously unrecognized aminopeptidase activity but no carboxy- or endopeptidase activity. These findings were corroborated by site-directed mutagenesis and identification of bestatin as a potent inhibitor of the enzyme. Analysis of NAALADL1 gene expression at both the mRNA and protein levels revealed the small intestine as the major site of protein expression and points toward extensive alternative splicing of the NAALADL1 gene transcript. Taken together, our data imply that the NAALADL1 gene product's primary physiological function is associated with the final stages of protein/peptide digestion and absorption in the human digestive system. Based on these results, we suggest a new name for this enzyme: human ileal aminopeptidase (HILAP).

Structural and biochemical characterization of the folyl-poly-γ-l-glutamate hydrolyzing activity of human glutamate carboxypeptidase II.

In addition to its well-characterized role in the central nervous system, human glutamate carboxypeptidase II (GCPII; Uniprot ID Q04609) acts as a folate hydrolase in the small intestine, participating in the absorption of dietary polyglutamylated folates (folyl-n-γ-l-glutamic acid), which are the provitamin form of folic acid (also known as vitamin B9 ). Despite the role of GCPII as a folate hydrolase, nothing is known about the processing of polyglutamylated folates by GCPII at the structural or enzymological level. Moreover, many epidemiologic studies on the relationship of the naturally occurring His475Tyr polymorphism to folic acid status suggest that this polymorphism may be associated with several pathologies linked to impaired folate metabolism. In the present study, we report: (a) a series X-ray structures of complexes between a catalytically inactive GCPII mutant (Glu424Ala) and a panel of naturally occurring polyglutamylated folates; (b) the X-ray structure of the His475Tyr variant at a resolution of 1.83 Å; (c) the study of the recently identified arene-binding site of GCPII through mutagenesis (Arg463Leu, Arg511Leu and Trp541Ala), inhibitor binding and enzyme kinetics with polyglutamylated folates as substrates; and (d) a comparison of the thermal stabilities and folate-hydrolyzing activities of GCPII wild-type and His475Tyr variants. As a result, the crystallographic data reveal considerable details about the binding mode of polyglutamylated folates to GCPII, especially the engagement of the arene binding site in recognizing the folic acid moiety. Additionally, the combined structural and kinetic data suggest that GCPII wild-type and His475Tyr variant are functionally identical.

Structural characterization of P1'-diversified urea-based inhibitors of glutamate carboxypeptidase II.

Urea-based inhibitors of human glutamate carboxypeptidase II (GCPII) have advanced into clinical trials for imaging metastatic prostate cancer. In parallel efforts, agents with increased lipophilicity have been designed and evaluated for targeting GCPII residing within the neuraxis. Here we report the structural and computational characterization of six complexes between GCPII and P1'-diversified urea-based inhibitors that have the C-terminal glutamate replaced by more hydrophobic moieties. The X-ray structures are complemented by quantum mechanics calculations that provide a quantitative insight into the GCPII/inhibitor interactions. These data can be used for the rational design of novel glutamate-free GCPII inhibitors with tailored physicochemical properties.

Preclinical evaluation of human secretoglobin 3A2 in mouse models of lung development and fibrosis.

Secretoglobin (SCGB) 3A2 is a member of the SCGB gene superfamily of small secreted proteins, predominantly expressed in lung airways. We hypothesize that human SCGB3A2 may exhibit anti-inflammatory, growth factor, and antifibrotic activities and be of clinical utility. Recombinant human SCGB3A2 was expressed, purified, and biochemically characterized as a first step to its development as a therapeutic agent in clinical settings. Human SCGB3A2, as well as mouse SCGB3A2, readily formed a dimer in solution and exhibited novel phospholipase A2 inhibitory activity. This is the first demonstration of any quantitative biochemical measurement for the evaluation of SCGB3A2 protein. In the mouse as an experimental animal, human SCGB3A2 exhibited growth factor activity by promoting embryonic lung development in both ex vivo and in vivo systems and antifibrotic activity in the bleomycin-induced lung fibrosis model. The results suggested that human SCGB3A2 can function as a growth factor and an antifibrotic agent in humans. When SCGB3A2 was administered to pregnant female mice through the tail vein, the protein was detected in the dam's serum and lung, as well as the placenta, amniotic fluids, and embryonic lungs at 10 min postadministration, suggesting that SCGB3A2 readily crosses the placenta. The results warrant further development of recombinant SCGB3A2 as a therapeutic agent in treating patients suffering from lung diseases or preterm infants with respiratory distress.

Human ß-defensin-3 structure motifs that are important in CXCR4 antagonism.

Previously, we reported that human ß-defensin (hBD)-3 can both antagonize CXCR4 function on T cells and promote receptor internalization in the absence of activation. In the present study, we explored the important structural elements of hBD-3 that are involved in blocking CXCR4 activation by its natural ligand, stromal-derived factor 1α (SDF-1α; CXCL12). Results from site-directed mutagenesis studies suggest that the ability of hBD-3 to inhibit SDF-1α-CXCR4 interaction, as assayed either by blocking SDF-1 binding to CXCR4 or antagonizing SDF-1-induced Ca(2+) mobilization, is correlated with the presence of hBD-3 cysteine residues, specific surface-distributed cationic residues, and the electrostatic properties and availability of both hBD-3 termini. Specifically, hBD-3 activity against CXCR4 is reduced by: (a) replacing all six cysteines; (b) replacing the cationic residues with acidic ones in the N-terminus and C- terminus; (c) removal of the first 10 N-terminal residues; and (d) replacing the surface-exposed basic residues Lys8, Lys32 and Arg36 with neutral ones. The hBD-3-CXCR4 interaction has potentially wide-ranging implications for HIV-related biology, as well as for a host of CXCR4-dependent activities, including hematopoiesis, neurogenesis, angiogenesis, carcinogenesis, and immune cell trafficking. CXCR4 is highly expressed on T cells, monocytes, and epithelial cells. Therefore, understanding the structure-function relationship between hBD-3 and CXCR4 that accounts for the antagonistic interaction between the two molecules may provide new insights into HIV/highly active antiretroviral therapy-related pathology, as well as novel insights into the interaction between innate and adaptive immunity at mucosal sites.