Alexei Gorelik

Structural Basis of Carbohydrate Recognition by Calreticulin

The calnexin cycle is a process by which glycosylated proteins are subjected to folding cycles in the endoplasmic reticulum lumen via binding to the membrane protein calnexin (CNX) or to its soluble homolog calreticulin (CRT). CNX and CRT specifically recognize monoglucosylated Glc1Man9GlcNAc2 glycans, but the structural determinants underlying this specificity are unknown. Here, we report a 1.95-Å crystal structure of the CRT lectin domain in complex with the tetrasaccharide α-Glc-(1→3)-α-Man-(1→2)-α-Man-(1→2)-Man. The tetrasaccharide binds to a long channel on CRT formed by a concave β-sheet. All four sugar moieties are engaged in the protein binding via an extensive network of hydrogen bonds and hydrophobic contacts. The structure explains the requirement for glucose at the nonreducing end of the carbohydrate; the oxygen O2 of glucose perfectly fits to a pocket formed by CRT side chains while forming direct hydrogen bonds with the carbonyl of Gly124 and the side chain of Lys111. The structure also explains a requirement for the Cys105–Cys137 disulfide bond in CRT/CNX for efficient carbohydrate binding. The Cys105–Cys137 disulfide bond is involved in intimate contacts with the third and fourth sugar moieties of the Glc1Man3 tetrasaccharide. Finally, the structure rationalizes previous mutagenesis of CRT and lays a structural groundwork for future studies of the role of CNX/CRT in diverse biological pathways.

Crystal structure of mammalian acid sphingomyelinase.

Acid sphingomyelinase (ASMase, ASM, SMPD1) converts sphingomyelin into ceramide, modulating membrane properties and signal transduction. Inactivating mutations in ASMase cause Niemann–Pick disease, and its inhibition is also beneficial in models of depression and cancer. To gain a better understanding of this critical therapeutic target, we determined crystal structures of mammalian ASMase in various conformations. The catalytic domain adopts a calcineurin-like fold with two zinc ions and a hydrophobic track leading to the active site. Strikingly, the membrane interacting saposin domain assumes either a closed globular conformation independent from the catalytic domain, or an open conformation, which establishes an interface with the catalytic domain essential for activity. Structural mapping of Niemann–Pick mutations reveals that most of them likely destabilize the proteins fold. This study sheds light on the molecular mechanism of ASMase function, and provides a platform for the rational development of ASMase inhibitors and therapeutic use of recombinant ASMase.

Crystal Structure of the Human Alkaline Sphingomyelinase Provides Insights into Substrate Recognition

Absorption of dietary sphingomyelin (SM) requires its initial degradation into ceramide, a process catalyzed by the intestinal enzyme alkaline sphingomyelinase (alk-SMase, NPP7, ENPP7). alk-SMase belongs to the nucleotide pyrophosphatase/phosphodiesterase (NPP) family, the members of which hydrolyze nucleoside phosphates, phospholipids, and other related molecules. NPP7 is the only paralog that can cleave SM, and its activity requires the presence of bile salts, a class of physiological anionic detergents. To elucidate the mechanism of substrate recognition, we determined the crystal structure of human alk-SMase in complex with phosphocholine, a reaction product. Although the overall fold and catalytic center are conserved relative to other NPPs, alk-SMase recognizes the choline moiety of its substrates via an NPP7-specific aromatic box composed of tyrosine residues. Mutational analysis and enzymatic activity assays identified features on the surface of the protein—a cationic patch and a unique hydrophobic loop—that are essential for accessing SM in bile salt micelles. These results shed new light on substrate specificity determinants within the NPP enzyme family.

Structural Basis for Nucleotide Hydrolysis by the Acid Sphingomyelinase-like Phosphodiesterase SMPDL3A.

Sphingomyelin phosphodiesterase, acid-like 3A (SMPDL3A) is a member of a small family of proteins founded by the well characterized lysosomal enzyme, acid sphingomyelinase (ASMase). ASMase converts sphingomyelin into the signaling lipid, ceramide. It was recently discovered that, in contrast to ASMase, SMPDL3A is inactive against sphingomyelin and, surprisingly, can instead hydrolyze nucleoside diphosphates and triphosphates, which may play a role in purinergic signaling. As none of the ASMase-like proteins has been structurally characterized to date, the molecular basis for their substrate preferences is unknown. Here we report crystal structures of murine SMPDL3A, which represent the first structures of an ASMase-like protein. The catalytic domain consists of a central mixed β-sandwich surrounded by α-helices. Additionally, SMPDL3A possesses a unique C-terminal domain formed from a cluster of four α-helices that appears to distinguish this protein family from other phosphoesterases. We show that SMDPL3A is a di-zinc-dependent enzyme with an active site configuration that suggests a mechanism of phosphodiester hydrolysis by a metal-activated water molecule and protonation of the leaving group by a histidine residue. Co-crystal structures of SMPDL3A with AMP and α,β-methylene ADP (AMPCP) reveal that the substrate binding site accommodates nucleotides by establishing interactions with their base, sugar, and phosphate moieties, with the latter the major contributor to binding affinity. Our study provides the structural basis for SMPDL3A substrate specificity and sheds new light on the function of ASMase-like proteins.

A key tyrosine substitution restricts nucleotide hydrolysis by the ectoenzyme NPP5

The ecto‐nucleotide pyrophosphatase/phosphodiesterase (NPP) family of proteins mediates purinergic signaling by degrading extracellular nucleotides and also participates in phospholipid metabolism. NPP5 (ENPP5) is the least characterized member of this group and its specific role is unknown. This enzyme does not display activity on certain nucleotides and on other typical NPP substrates. In order to gain insights into its function, we determined the crystal structure of human and murine NPP5. Structural comparison with close homologs revealed a key phenylalanine to tyrosine substitution that prevents efficient hydrolysis of nucleotide diphosphates and triphosphates; reversal of this mutation enabled degradation of these molecules. Interestingly, NPP5 is able to cleave nicotinamide adenine dinucleotide (NAD), suggesting a potential role of this enzyme in NAD‐based neurotransmission. An NPP5‐specific metal binding motif is found adjacent to the active site, although its significance is unclear. These findings expand our understanding of substrate specificity within the NPP family.

Crystal structure of the mammalian lipopolysaccharide detoxifier

Significance LPS is the major bacterial molecule recognized by the human innate immune system. It elicits a strong inflammatory response followed by a state of immune tolerance. The human enzyme acyloxyacyl hydrolase (AOAH) then detoxifies LPS to reestablish sensitivity for subsequent infections. We determined the 3D structure of AOAH to better understand its function. The enzyme binds to LPS via a hydrophobic surface and contains a hydrophobic tunnel into which one fatty acyl chain of the LPS fits to be cleaved off. AOAH also interacts with the phosphate groups of LPS but not its saccharide portion. Additionally, we report an unexpected calcium-binding domain in this enzyme. LPS is a potent bacterial endotoxin that triggers the innate immune system. Proper recognition of LPS by pattern-recognition receptors requires a full complement of typically six acyl chains in the lipid portion. Acyloxyacyl hydrolase (AOAH) is a host enzyme that removes secondary (acyloxyacyl-linked) fatty acids from LPS, rendering it immunologically inert. This activity is critical for recovery from immune tolerance that follows Gram-negative infection. To understand the molecular mechanism of AOAH function, we determined its crystal structure and its complex with LPS. The substrate’s lipid moiety is accommodated in a large hydrophobic pocket formed by the saposin and catalytic domains with a secondary acyl chain inserted into a narrow lateral hydrophobic tunnel at the active site. The enzyme establishes dispensable contacts with the phosphate groups of LPS but does not interact with its oligosaccharide portion. Proteolytic processing allows movement of an amphipathic helix possibly involved in substrate access at membranes.

Crystal Structure of the Acid Sphingomyelinase-Like Phosphodiesterase SMPDL3B Provides Insights into Determinants of Substrate Specificity

The enzyme acid sphingomyelinase-like phosphodiesterase 3B (SMPDL3B) was shown to act as a negative regulator of innate immune signaling, affecting cellular lipid composition and membrane fluidity. Furthermore, several reports identified this enzyme as an off target of the therapeutic antibody rituximab, with implications in kidney disorders. However, structural information for this protein is lacking. Here we present the high resolution crystal structure of murine SMPDL3B, which reveals a substrate binding site strikingly different from its paralogs. The active site is located in a narrow boot-shaped cavity. We identify a unique loop near the active site that appears to impose size constraints on incoming substrates. A structure in complex with phosphocholine indicates that the protein recognizes this head group via an aromatic box, a typical choline-binding motif. Although a potential substrate for SMPDL3B is sphingomyelin, we identify other possible substrates such as CDP-choline, ATP, and ADP. Functional experiments employing structure-guided mutagenesis in macrophages highlight amino acid residues potentially involved in recognition of endogenous substrates. Our study is an important step toward elucidating the specific function of this poorly characterized enzyme.

Structural basis for nucleotide recognition by the ectoenzyme CD203c.

The ecto‐nucleotide pyrophosphatase/phosphodiesterase (NPP) enzyme family modulates purinergic signaling by degrading extracellular nucleotides. CD203c (NPP3, ENPP3) regulates the inflammatory response of basophils via ATP hydrolysis and is a marker for allergen sensitivity on the surface of these cells. Multiple other roles and substrates have also been proposed for this protein. In order to gain insight into its molecular functions, we determined the crystal structure of human NPP3 as well as its complex with an ATP analog. The enzyme exhibits little preference for nucleobase type, and forms specific contacts with the alpha and beta phosphate groups of its ligands. Dimerization of the protein does not affect its catalytic activity. These findings expand our understanding of substrate recognition within the NPP family.

Crystal structure of saposin D in an open conformation.

Saposins are accessory proteins that aid in the degradation of sphingolipids by hydrolytic enzymes. Their structure usually comprises four α-helices arranged in various conformations including an open, V-shaped form that is generally associated with the ability to interact with membranes and/or enzymes to accentuate activity. Saposin D is required by the lysosomal hydrolase, acid ceramidase, which breaks down ceramide into sphingosine and free fatty acid, to display optimal activity. The structure of saposin D was previously determined in an inactive conformation, revealing a monomeric, closed and compact form. Here, we present the crystal structure of the open, V-shaped form of saposin D. The overall shape is similar to the open conformation found in other saposins with slight differences in the angles between the α-helices. The structure forms a dimer that serves to stabilize the hydrophobic surface exposed in the open form, which results in an internal, hydrophobic cavity that could be used to carry extracted membrane lipids.