Structure-guided discovery of aminopeptidase ERAP1 variants capable of processing antigens with novel PC anchor specificities.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) belongs to the oxytocinase subfamily of M1 aminopeptidases (M1APs), which are a diverse family of metalloenzymes involved in a wide range of functions and have been implicated in various chronic and infectious diseases of humans. ERAP1 trims antigenic precursors into correct sizes (8-10 residues long) for Major Histocompatibility Complex (MHC) presentation, by a unique molecular ruler mechanism in which it makes concurrent bindings to substrate N- and C-termini. We have previously determined four crystal structures of ERAP1 C-terminal regulatory domain (termed ERAP1_C domain) in complex with peptide carboxyl (PC)-ends that carry various anchor residues, and identified a specificity subsite for recognizing the PC anchor side chain, denoted as the SC subsite to follow the conventional notations: S1 site for P1, S2 site for P2, and so forth. In this study, we report studies on structure-guided mutational and hydrolysis kinetics, and peptide trimming assays to further examine the functional roles of this SC subsite. Most strikingly, a point mutation V737R results in a change of substrate preference from a hydrophobic to a negatively charged PC anchor residue; the latter is presumed to be a poor substrate for WT ERAP1. These studies validate the crystallographic observations that this SC subsite is directly involved in binding and recognition of the substrate PC anchor and presents a potential target to modulate MHC-restricted immunopeptidomes.

ERAP1 binds peptide C-termini of different sequences and/or lengths by a common recognition mechanism.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) plays a key role in controlling the immunopeptidomes available for presentation by MHC (major histocompatibility complex) molecules, thus influences immunodominance and cell-mediated immunity. It carries out this critical function by a unique molecular ruler mechanism that trims antigenic precursors in a peptide-length and sequence dependent manner. Acting as a molecular ruler, ERAP1 is capable of concurrently binding antigen peptide N- and C-termini by its N-terminal catalytic and C-terminal regulatory domains, respectively. As such ERAP1 can not only monitor substrate's lengths, but also exhibit a degree of sequence specificity at substrates' N- and C-termini. On the other hand, it also allows certain sequence and length flexibility in the middle part of peptide substrates that is critical for shaping MHC restricted immunopeptidomes. Here we report structural and biochemical studies to understand the molecular details on how ERAP1 can accommodate side chains of different anchoring residues at the substrate's C-terminus. We also examine how ERAP1 can accommodate antigen peptide precursors with length flexibility. Based on two newly determined complex structures, we find that ERAP1 binds the C-termini of peptides similarly even with different substrate sequences and/or lengths, by utilizing the same hydrophobic specificity pocket to accommodate peptides with either a Phe or Leu as the C-terminal anchor residue. In addition, SPR (surface plasmon resonance) binding analyses in solution further confirm the biological significance of these peptide-ERAP1 interactions. Similar to the binding mode of MHC-I molecules, ERAP1 accommodates for antigenic peptide length difference by allowing the peptide middle part to kink or bulge at the middle of its substrate binding cleft. This explains how SNP coded variants located at the middle of ERAP1 substrate binding cleft would influence the antigen pool and an individual's susceptibility to diseases.

The T99K variant of glycosylasparaginase shows a new structural mechanism of the genetic disease aspartylglucosaminuria.

Aspartylglucosaminuria (AGU) is an inherited disease caused by mutations in a lysosomal amidase called aspartylglucosaminidase (AGA) or glycosylasparaginase (GA). This disorder results in an accumulation of glycoasparagines in the lysosomes of virtually all cell types, with severe clinical symptoms affecting the central nervous system, skeletal abnormalities, and connective tissue lesions. GA is synthesized as a single-chain precursor that requires an intramolecular autoprocessing to form a mature amidase. Previously, we showed that a Canadian AGU mutation disrupts this obligatory intramolecular autoprocessing with the enzyme trapped as an inactive precursor. Here, we report biochemical and structural characterization of a model enzyme corresponding to a new American AGU allele, the T99K variant. Unlike other variants with known 3D structures, this T99K model enzyme still has autoprocessing capacity to generate a mature form. However, its amidase activity to digest glycoasparagines remains low, consistent with its association with AGU. We have determined a 1.5-Å-resolution structure of this new AGU model enzyme and built an enzyme-substrate complex to provide a structural basis to analyze the negative effects of the T99K point mutation on KM and kcat of the amidase. It appears that a "molecular clamp" capable of fixing local disorders at the dimer interface might be able to rescue the deficiency of this new AGU variant.

Crystal structure of a polypeptide's C-terminus in complex with the regulatory domain of ER aminopeptidase 1.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is involved in the final processing of peptide precursors to generate the N-termini of MHC class I-restricted epitopes. ERAP1 thus influences immunodominance and cytotoxic immune responses by controlling the peptide repertoire available for cell surface presentation by MHC molecules. To enable this critical role in antigen processing, ERAP1 trims peptides by a unique molecular ruler mechanism that turns on/off hydrolysis activity in a peptide-length and -sequence dependent manner. Thus unlike other aminopeptidases, ERAP1 could recognize both the N- and C-termini of peptides in order to read the substrate's length. To exemplify and validate this molecular ruler mechanism, we have carried out crystallographic studies on molecular recognition of antigenic peptide's C-terminus by ERAP1. In this report, we have determined a 2.8Å-resolution crystal structure of an intermolecular complex between the ERAP1 regulatory domain and a natural epitope's C-terminus displayed in a fusion protein. It reveals the structural details of peptide's C-termini recognition by ERAP1. ERAP1 uses specificity pockets on the regulatory domain to bind the peptide's carboxyl end and side chain of the C-terminal anchoring residue. At the same time, flexibility in length and sequence at the middle of peptides is accommodated by a kink with minimal interactions with ERAP1.

Structural insights into the molecular ruler mechanism of the endoplasmic reticulum aminopeptidase ERAP1.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an essential component of the immune system, because it trims peptide precursors and generates the N--restricted epitopes. To examine ERAP1's unique properties of length- and sequence-dependent processing of antigen precursors, we report a 2.3 Å resolution complex structure of the ERAP1 regulatory domain. Our study reveals a binding conformation of ERAP1 to the carboxyl terminus of a peptide, and thus provides direct evidence for the molecular ruler mechanism.

Leucine motif-dependent tyrosine autophosphorylation of type III receptor tyrosine kinases.

Activation loop tyrosine autophosphorylation is an essential requirement for full kinase activation of receptor tyrosine kinases (RTKs). However, mechanisms involved are not fully understood. In general, kinase domains of RTKs are folded into two main lobes, NH2- and COOH-terminal lobes. The COOH-terminal lobe of vascular endothelial growth factor receptor-2 (VEGFR-2) is folded into seven alpha-helices (alphaD-alphaI). In the studies presented here we demonstrate that leucine residues of helix I (alphaI) regulate tyrosine autophosphorylation and phosphotransferase activity of VEGFR-2. The presence of leucines 1158, 1161, and 1162 are essential for tyrosine autophosphorylation and kinase activation of VEGFR-2 and are involved in helix-helix packing via hydrophobic interactions. The presence of leucine 1158 is critical for kinase activation of VEGFR-2 and appears to interact with alphaE, alphaF, alphaH, and beta7. The analogous residue, leucine 957 on platelet-derived growth factor receptor-beta and leucine 910 on colony stimulating factor-1R are also found to be critical for tyrosine autophosphorylation of these receptors. Leucines 1161 and 1162 are also involved in helix-helix packing but they play a less critical role in VEGFR-2 activation. Thus, we conclude that leucine motif-mediated helix-helix interactions are critical for kinase regulation of type III RTKs. This mechanism is likely to be shared with other kinases and might provide a basis for the design of a novel class of tyrosine kinase inhibitors.

Two crystal forms of the restriction enzyme MspI-DNA complex show the same novel structure.

The crystal structure of the Type IIP restriction endonuclease MspI bound to DNA containing its cognate recognition sequence has been determined in both monoclinic and orthorhombic space groups. Significantly, these two independent crystal forms present an identical structure of a novel monomer-DNA complex, suggesting a functional role for this novel enzyme-DNA complex. In both crystals, MspI interacts with the CCGG DNA recognition sequence as a monomer, using an asymmetric mode of recognition by two different structural motifs in a single polypeptide. In the crystallographic asymmetric unit, the two DNA molecules in the two MspI-DNA complexes appear to stack with each other forming an end-to-end pseudo-continuous 19-mer duplex. They are primarily B-form and no major bends or kinks are observed. For DNA recognition, most of the specific contacts between the enzyme and the DNA are preserved in the orthorhombic structure compared with the monoclinic structure. A cation is observed near the catalytic center in the monoclinic structure at a position homologous to the 74/45 metal site of EcoRV, and the orthorhombic structure also shows signs of this same cation. However, the coordination ligands of the metal are somewhat different from those of the 74/45 metal site of EcoRV. Combined with structural information from other solved structures of Type II restriction enzymes, the possible relationship between the structures of the enzymes and their cleavage behaviors is discussed.

An asymmetric complex of restriction endonuclease MspI on its palindromic DNA recognition site.

Most well-known restriction endonucleases recognize palindromic DNA sequences and are classified as Type IIP. Due to the recognition and cleavage symmetry, Type IIP enzymes are usually found to act as homodimers in forming 2-fold symmetric enzyme-DNA complexes. Here we report an asymmetric complex of the Type IIP restriction enzyme MspI in complex with its cognate recognition sequence. Unlike any other Type IIP enzyme reported to date, an MspI monomer and not a dimer binds to a palindromic DNA sequence. The enzyme makes specific contacts with all 4 base pairs in the recognition sequence, by six direct and five water-mediated hydrogen bonds and numerous van der Waal contacts. This MspI-DNA structure represents the first example of asymmetric recognition of a palindromic DNA sequence by two different structural motifs in one polypeptide. A few possible pathways are discussed for MspI to cut both strands of DNA, either as a monomer or dimer.

Two-step dimerization for autoproteolysis to activate glycosylasparaginase.

Glycosylasparaginase (GA) is an amidase and belongs to a novel family of N-terminal nucleophile hydrolases that use a similar autoproteolytic processing mechanism to generate a mature/active enzyme from a single chain protein precursor. From bacteria to eukaryotes, GAs are conserved in primary sequences, tertiary structures, and activation of amidase activity by intramolecular autoproteolysis. An evolutionarily conserved His-Asp-Thr sequence is cleaved to generate a newly exposed N-terminal threonine, which plays a central role in both autoproteolysis and in its amidase activity. We have recently determined the crystal structure of the bacterial GA precursor at 1.9-A resolution, which reveals a highly distorted and energetically unfavorable conformation at the scissile peptide bond. A mechanism of autoproteolysis via an N-O acyl shift was proposed to relieve these conformational strains. However, it is not understood how the polypeptide chain distortion was generated and preserved during the folding of GA to trigger autoproteolysis. An obstacle to our understanding of GA autoproteolysis is the uncertainty concerning its quaternary structure in solution. Here we have revisited this question and show that GA forms dimers in solution. Mutants with alterations at the dimer interface cannot form dimers and are impaired in the autoproteolytic activation. This suggests that dimerization of GA plays an essential role in autoproteolysis to activate the amidase activity. Comparison of the melting temperatures of GA dimers before and after autoproteolysis suggests two states of dimerization in the process of enzyme maturation. A two-step dimerization mechanism to trigger autoproteolysis is proposed to accommodate the data presented here as well as those in the literature.