Sebastian Springer

A primer on vesicle budding

A basic scheme of transport vesicle formation is beginning to emerge (Figure 1Figure 1). Its central feature is the priming complex: an association of a small GTPase, a primer protein, and one or several other subunits of the vesicular coat, including a GTPase-activating protein. Formation of this complex is coat- and membrane-specific due to the restricted overlapping cellular distributions of primer proteins and GTPases in their GTP state; functionally essential vesicle proteins may prove crucial as primers for its nucleation; in the priming complex, coat proteins that recognize primers may change their conformation; and finally, the priming complex may decay after the GTPase is stimulated to hydrolyze GTP, giving rise to free GTPase and a polymeric coat on the membrane. The specific predictions that arise from this model may also apply to the uptake of membrane proteins into cellular transport vesicles with different coats, such as clathrin at the trans-Golgi network and the plasma membrane, or AP-3 in trans-Golgi network to endosome traffic.‡To whom correspondence should be addressed (e-mail: schekman@uclink4.berkeley.edu).

Structure of the Sec23p/24p and Sec13p/31p complexes of COPII

COPII-coated vesicles carry proteins from the endoplasmic reticulum to the Golgi complex. This vesicular transport can be reconstituted by using three cytosolic components containing five proteins: the small GTPase Sar1p, the Sec23p/24p complex, and the Sec13p/Sec31p complex. We have used a combination of biochemistry and electron microscopy to investigate the molecular organization and structure of Sec23p/24p and Sec13p/31p complexes. The three-dimensional reconstruction of Sec23p/24p reveals that it has a bone-shaped structure, (17 nm in length), composed of two similar globular domains, one corresponding to Sec23p and the other to Sec24p. Sec13p/31p is a heterotetramer composed of two copies of Sec13p and two copies of Sec31p. It has an elongated shape, is 28–30 nm in length, and contains five consecutive globular domains linked by relatively flexible joints. Putting together the architecture of these Sec complexes with the interactions between their subunits and the appearance of the coat in COPII-coated vesicles, we present a model for COPII coat organization.

Crystal structures of two H-2Db/glycopeptide complexes suggest a molecular basis for CTL cross-reactivity.

Two synthetic O-GlcNAc-bearing peptides that elicit H-2Db-restricted glycopeptide-specific cytotoxic T cells (CTL) have been shown to display nonreciprocal patterns of cross-reactivity. Here, we present the crystal structures of the H-2Db glycopeptide complexes to 2.85 A resolution or better. In both cases, the glycan is solvent exposed and available for direct recognition by the T cell receptor (TCR). We have modeled the complex formed between the MHC-glycopeptide complexes and their respective TCRs, showing that a single saccharide residue can be accommodated in the standard TCR-MHC geometry. The models also reveal a possible molecular basis for the observed cross-reactivity patterns of the CTL clones, which appear to be influenced by the length of the CDR3 loop and the nature of the immunizing ligand.

Controlled intracellular release of peptides from microcapsules enhances antigen presentation on MHC class I molecules.

To understand the time course of action of any small molecule inside a single cell, one would deposit a defined amount inside the cell and initiate its activity at a defined moment. An elegant way to achieve this is to encapsulate the molecule in a micrometer-sized reservoir, introduce it into a cell, remotely open its wall by a laser pulse, and then follow the biological response by microscopy. The validity of this approach is validated here using microcapsules with defined walls that are doped with metallic nanoparticles so as to enable them to be opened with an infrared laser. The capsules are loaded with a fluorescent antigenic peptide and introduced into mammalian cultured cells where, upon laser-induced release, the peptide binds to major histocompatibility complex (MHC) class I proteins and elicits their cell surface transport. The concept of releasing a drug inside a cell and following its action is applicable to many problems in cell biology and medicine.

Tapasin and other chaperones: models of the MHC class I loading complex

Abstract MHC (major histocompatibility complex) class I molecules bind intracellular virus-derived peptides in the endoplasmic reticulum (ER) and present them at the cell surface to cytotoxic T lymphocytes. Peptide-free class I molecules at the cell surface, however, could lead to aberrant T cell killing. Therefore, cells ensure that class I molecules bind high-affinity ligand peptides in the ER, and restrict the export of empty class I molecules to the Golgi apparatus. For both of these safeguard mechanisms, the MHC class I loading complex (which consists of the peptide transporter TAP, the chaperones tapasin and calreticulin, and the protein disulfide isomerase ERp57) plays a central role. This article reviews the actions of accessory proteins in the biogenesis of class I molecules, specifically the functions of the loading complex in high-affinity peptide binding and localization of class I molecules, and the known connections between these two regulatory mechanisms. It introduces new models for the mode of action of tapasin, the role of the class I loading complex in peptide editing, and the intracellular localization of class I molecules.

Calreticulin‐dependent recycling in the early secretory pathway mediates optimal peptide loading of MHC class I molecules

Calreticulin is a lectin chaperone of the endoplasmic reticulum (ER). In calreticulin‐deficient cells, major histocompatibility complex (MHC) class I molecules travel to the cell surface in association with a sub‐optimal peptide load. Here, we show that calreticulin exits the ER to accumulate in the ER–Golgi intermediate compartment (ERGIC) and the cis‐Golgi, together with sub‐optimally loaded class I molecules. Calreticulin that lacks its C‐terminal KDEL retrieval sequence assembles with the peptide‐loading complex but neither retrieves sub‐optimally loaded class I molecules from the cis‐Golgi to the ER, nor supports optimal peptide loading. Our study, to the best of our knowledge, demonstrates for the first time a functional role of intracellular transport in the optimal loading of MHC class I molecules with antigenic peptide.

Flexibility of the MHC class II peptide binding cleft in the bound, partially filled, and empty states: A molecular dynamics simulation study

Major histocompatibility (MHC) Class II cell surface proteins present antigenic peptides to the immune system. Class II structures in complex with peptides but not in the absence of peptide are known. Comparative molecular dynamics (MD) simulations of a Class II protein (HLA-DR3) with and without CLIP (invariant chain-associated protein) peptide were performed starting from the CLIP-bound crystal structure. Depending on the protonation of acidic residues in the P6 peptide-binding pocket the simulations stayed overall close to the start structure. The simulations without CLIP showed larger conformational fluctuations especially of alpha-helices flanking the binding cleft. Largest fluctuations without CLIP were observed in a helical segment near the peptide C-terminus binding region matching a segment recognized by antibodies specific for empty Class II proteins. Simulations on a Val86Tyr mutation that fills the peptide N-terminus binding P1 pocket or of a complex with a CLIP fragment (dipeptide) bound to P1 showed an unexpected long range effect. In both simulations the mobility not only of P1 but also of the entire binding cleft was reduced compared to simulations without CLIP. It correlates with the experimental finding that the CLIP fragment binding to P1 is sufficient to prevent antibody recognition specific for the empty form at a site distant from P1. The results suggest a mechanism how a local binding event of small peptides or of an exchange factor near P1 may promote peptide binding and exchange through a long range stabilization of the whole binding cleft in a receptive (near bound) conformation.

Comparative molecular dynamics analysis of tapasin-dependent and -independent MHC class I alleles

MHC class I molecules load antigenic peptides in the endoplasmic reticulum and present them at the cell surface. Efficiency of peptide loading depends on the class I allele and can involve interaction with tapasin and other proteins of the loading complex. Allele HLA‐B*4402 (Asp at position 116) depends on tapasin for efficient peptide loading, whereas HLA‐B*4405 (identical to B*4402 except for Tyr116) can efficiently load peptides in the absence of tapasin. Both alleles adopt very similar structures in the presence of the same peptide. Comparative unrestrained molecular dynamics simulations on the α1/α2 peptide binding domains performed in the presence of bound peptides resulted in structures in close agreement with experiments for both alleles. In the absence of peptides, allele‐specific conformational changes occurred in the first segment of the α2‐helix that flanks the peptide C‐terminal binding region (F‐pocket) and contacts residue 116. This segment is also close to the proposed tapasin contact region. For B*4402, a shift toward an altered F‐pocket structure deviating significantly from the bound form was observed. Subsequent free energy simulations on induced F‐pocket opening in B*4402 confirmed a conformation that deviated significantly from the bound structure. For B*4405, a free energy minimum close to the bound structure was found. The simulations suggest that B*4405 has a greater tendency to adopt a peptide receptive conformation in the absence of peptide, allowing tapasin‐independent peptide loading. A possible role of tapasin could be the stabilization of a peptide‐receptive class I conformation for HLA‐B*4402 and other tapasin‐dependent alleles.

Tapasin edits peptides on MHC class I molecules by accelerating peptide exchange

The endoplasmic reticulum (ER) protein tapasin is essential for the loading of high‐affinity peptides onto MHC class I molecules. It mediates peptide editing, i.e. the binding of peptides of successively higher affinity until class I molecules pass ER quality control and exit to the cell surface. The molecular mechanism of action of tapasin is unknown. We describe here the reconstitution of tapasin‐mediated peptide editing on class I molecules in the lumen of microsomal membranes. We find that in a competitive situation between high‐ and low‐affinity peptides, tapasin mediates the binding of the high‐affinity peptide to class I by accelerating the dissociation of the peptide from an unstable intermediate of the binding reaction.

Differential tapasin dependence of MHC class I molecules correlates with conformational changes upon peptide dissociation: a molecular dynamics simulation study.

Efficiency of peptide loading to MHC class I molecules in the endoplasmic reticulum is allele specific and can involve interaction with tapasin and other proteins. Allele HLA-B 4,402 depends on tapasin whereas HLA-B 4,405 (Tyr116 instead of Asp in B 4,402) can efficiently load peptides without tapasin. Both alleles adopt very similar structures in the presence of the same peptide. Molecular dynamics simulations on peptide termini dissociation from the alpha(1)/alpha(2) binding domains were used to characterize structural and free energy changes. The magnitude of the calculated free energy change and the shape of the free energy curve vs. distance for induced peptide C terminus dissociation differed for B 4,405 compared to B 4,402. Structural changes during C terminus dissociation occurred mainly in the first segment of the alpha(2)-helix that flanks the peptide C terminus binding region (F pocket) and contacts residue 116. This segment is also close to the proposed tapasin contact region. For B 4402, a stable shift towards an altered open F pocket structure deviating significantly from the bound form was observed. In contrast, B 4405 showed only a transient opening of the F pocket followed by relaxation towards a structure close to the bound (receptive) form upon C terminus dissociation. The greater tendency for a peptide-receptive conformation in the absence of peptide combined with more long-range interactions with the peptide C terminus facilitates peptide binding to B 4405 and correlates with the tapasin-independence of this allele. A possible role of tapasin in case of HLA-B 4402 and other tapasin-dependent alleles could be the stabilization of a peptide-receptive class I conformation.