PDB-9pkv: HU-38 Fab with PRAME pMHC

Basic information

• Entry: Database: PDB / ID: 9pkv
• Title: HU-38 Fab with PRAME pMHC

Components

• Beta-2-microglobulin
• HU-38 Heavy Chain
• HU-38 Light Chain
• MHC class I antigen
• PRAME peptide
• VHH antibody

• Keywords: PEPTIDE BINDING PROTEIN/IMMUNE SYSTEM / PRAME / pMHC / PEPTIDE BINDING PROTEIN / PEPTIDE BINDING PROTEIN-IMMUNE SYSTEM complex

Function / homology

Function:

negative regulation of receptor binding / early endosome lumen / Nef mediated downregulation of MHC class I complex cell surface expression / DAP12 interactions / transferrin transport / cellular response to iron ion / Endosomal/Vacuolar pathway / Antigen Presentation: Folding, assembly and peptide loading of class I MHC / peptide antigen assembly with MHC class II protein complex / cellular response to iron(III) ion / MHC class II protein complex / negative regulation of forebrain neuron differentiation / antigen processing and presentation of exogenous protein antigen via MHC class Ib, TAP-dependent / ER to Golgi transport vesicle membrane / peptide antigen assembly with MHC class I protein complex / regulation of erythrocyte differentiation / regulation of iron ion transport / HFE-transferrin receptor complex / response to molecule of bacterial origin / MHC class I peptide loading complex / T cell mediated cytotoxicity / positive regulation of T cell cytokine production / antigen processing and presentation of endogenous peptide antigen via MHC class I / antigen processing and presentation of exogenous peptide antigen via MHC class II / positive regulation of immune response / MHC class I protein complex / positive regulation of T cell activation / peptide antigen binding / positive regulation of receptor-mediated endocytosis / negative regulation of neurogenesis / cellular response to nicotine / positive regulation of T cell mediated cytotoxicity / multicellular organismal-level iron ion homeostasis / Modulation by Mtb of host immune system / specific granule lumen / phagocytic vesicle membrane / recycling endosome membrane / Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell / Interferon gamma signaling / negative regulation of epithelial cell proliferation / MHC class II protein complex binding / late endosome membrane / sensory perception of smell / positive regulation of cellular senescence / tertiary granule lumen / DAP12 signaling / T cell differentiation in thymus / ER-Phagosome pathway / negative regulation of neuron projection development / protein refolding / early endosome membrane / protein homotetramerization / amyloid fibril formation / intracellular iron ion homeostasis / learning or memory / Amyloid fiber formation / endoplasmic reticulum lumen / Golgi membrane / lysosomal membrane / external side of plasma membrane / focal adhesion / Neutrophil degranulation / SARS-CoV-2 activates/modulates innate and adaptive immune responses / structural molecule activity / endoplasmic reticulum / Golgi apparatus / protein homodimerization activity / extracellular space / extracellular exosome / extracellular region / identical protein binding / membrane / plasma membrane / cytosol

Domain/homology:

MHC class I alpha chain, alpha1 alpha2 domains / Class I Histocompatibility antigen, domains alpha 1 and 2 / Beta-2-Microglobulin / : / MHC class I-like antigen recognition-like / MHC class I-like antigen recognition-like superfamily / MHC classes I/II-like antigen recognition protein / : / Immunoglobulin/major histocompatibility complex, conserved site / Immunoglobulins and major histocompatibility complex proteins signature. / Immunoglobulin C-Type / Immunoglobulin C1-set / Immunoglobulin C1-set domain / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold

Component:

Beta-2-microglobulin / MHC class I antigen

• Biological species: Homo sapiens (human); Lama glama (llama); Mus musculus (house mouse)
• Method: ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.72 Å
• Authors: Mortenson, D.E. / Yu, X.

Funding support

1items

Organization	Grant number	Country
Not funded

• Citation: Journal: MAbs / Year: 2025; Title: A highly selective TCR-mimic antibody reveals unexpected mechanisms of HBV peptide-MHC recognition and previously unknown target biology.; Authors: Shahzada Khan / Jeremy Lum / Heather Stephenson / Pawan Bir Kohli / David Mortenson / Dhivya Ramakrishnan / Magdeleine Hung / Sheng Ding / Elbert Seto / Sabrina Lu / Randy Yen / Debi Jin / Brian Lee / Sheila Clancy / Nicole Schirle Oakdale / Nikolai Novikov / Don Kang / Ruidong Li / David Pan / Rutwij Dave / Eric Lansdon / Simon P Fletcher / Abhishek V Garg / Nathan Thomsen / Scott Balsitis / ; Abstract: Curative therapies for chronic hepatitis B virus infection (CHB) are needed, and T-cell redirection is a promising approach, with peptide-MHC complexes (pMHC) being attractive targets. HBV core peptide (C18, 10-mer) presented by HLA-A*02:01 (C18-MHC) has two major variants (C18-V or C18-I, differing in the C-terminal residue), both of which are known to be targeted by CD8 T cells in HBV-infected individuals. Through an extensive screening campaign, we identified a highly selective anti-C18-MHC antibody clone MUR35. A MUR35-based T-cell engager (TCE) potently killed HBV-infected hepatocytes but had no activity on uninfected hepatocytes, on other HBV-negative cell types or on host peptides with sequence similarity to C18. Crystal structures of MUR35 bound to both C18-I- and C18-V-MHC revealed a unique binding mode with contacts mediated exclusively by the light chain complementarity-determining regions (CDRs), suggesting that high specificity is achievable without a typical T-cell receptor-like binding mode involving both heavy and light chain CDRs. Although MUR35 exhibits similar binding affinity and structural contacts with C18-V and C18-I, TCE killing was only detected on hepatocytes producing C18-V. To better understand the cause of this discrepancy, we conducted a quantitative proteomics study in an HBV-infected humanized mouse model and found that C18-V was expressed at approximately 300 copies/cell, while C18-I expression was below the limit of detection. Unexpectedly, the proteomics studies revealed that previously unreported 9-mers missing the N-terminal phenylalanine of C18-I and -V were expressed at an average of 508 and 142 copies/cell, respectively, and therefore could be alternative targets for HBV pan genotypic coverage. Our data suggest unexpectedly large differences in antigen presentation efficiency between highly conservative amino acid substitutions in C18 peptide and reveal potentially novel HBV targets for future studies.

History

Deposition	Jul 14, 2025	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Oct 1, 2025	Provider: repository / Type: Initial release

Assembly

Deposited unit

A: MHC class I antigen

K: VHH antibody

P: PRAME peptide

H: HU-38 Heavy Chain

B: Beta-2-microglobulin

L: HU-38 Light Chain

Summary:

• 109 kDa, 6 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	109,448	6
Polymers	109,448	6
Non-polymers	0	0
Water	3,441	191

1

Summary:

• Idetical with deposited unit
• defined by author
• Evidence: electron microscopy, not applicable

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555		1

Components

Protein , 2 types, 2 molecules A B

#1: Protein

A MHC class I antigen

Details:

Mass: 34359.895 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HLA-A / Production host: Escherichia coli (E. coli) / References: UniProt: Q8WLS4

#5: Protein

B Beta-2-microglobulin

Details:

Mass: 11879.356 Da / Num. of mol.: 1 / Fragment: UNP residues 21-119
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: B2M, CDABP0092, HDCMA22P / Production host: Escherichia coli (E. coli) / References: UniProt: P61769

Antibody , 3 types, 3 molecules K H L

#2: Antibody

K VHH antibody

Details:

Mass: 13246.515 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Saccharomyces cerevisiae (brewer's yeast)

#4: Antibody

H HU-38 Heavy Chain

Details:

Mass: 25680.639 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): expi293 / Production host: Homo sapiens (human)

#6: Antibody

L HU-38 Light Chain

Details:

Mass: 23287.822 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): expi293 / Production host: Homo sapiens (human)

Protein/peptide / Non-polymers , 2 types, 192 molecules P

#3: Protein/peptide

P PRAME peptide

Details:

Mass: 994.209 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

#7: Water

ChemComp-HOH / water

Details:

Mass: 18.015 Da / Num. of mol.: 191 / Source method: isolated from a natural source / Formula: H₂O

Details

• Has ligand of interest: N
• Has protein modification: Y

Experimental details

Experiment

• Experiment: Method: ELECTRON MICROSCOPY
• EM experiment: Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

Sample preparation

• Component: Name: complex of HU38 Fab with PRAME pMHC and anti-Kappa VHH; Type: COMPLEX / Entity ID: #1-#6 / Source: MULTIPLE SOURCES
• Molecular weight: Experimental value: NO
• Source (natural): Organism: Homo sapiens (human)
• Source (recombinant): Organism: Escherichia coli (E. coli)
• Buffer solution: pH: 7.4
• Specimen: Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
• Vitrification: Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 10 K

Electron microscopy imaging

• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company
• Microscopy: Model: TFS KRIOS
• Electron gun: Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
• Electron lens: Mode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
• Image recording: Electron dose: 50 e/Ų / Film or detector model: GATAN K3 (6k x 4k)

Processing

EM software

ID	Name	Version	Category
1	cryoSPARC		particle selection
2	PHENIX	1.21_5207	model refinement
11	cryoSPARC		final Euler assignment
13	cryoSPARC		3D reconstruction

• CTF correction: Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
• 3D reconstruction: Resolution: 2.72 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 553793 / Symmetry type: POINT