Guennadi Kozlov

Structural proteomics of an archaeon.

A set of 424 nonmembrane proteins from Methanobacterium thermoautotrophicum were cloned, expressed and purified for structural studies. Of these, ∼20% were found to be suitable candidates for X-ray crystallographic or NMR spectroscopic analysis without further optimization of conditions, providing an estimate of the number of the most accessible structural targets in the proteome. A retrospective analysis of the experimental behavior of these proteins suggested some simple relations between sequence and solubility, implying that data bases of protein properties will be useful in optimizing high throughput strategies. Of the first 10 structures determined, several provided clues to biochemical functions that were not detectable from sequence analysis, and in many cases these putative functions could be readily confirmed by biochemical methods. This demonstrates that structural proteomics is feasible and can play a central role in functional genomics.

Structure of Parkin Reveals Mechanisms for Ubiquitin Ligase Activation

Parkin Enhanced? Inactivation of parkin, an E3 ubiquitin ligase, is responsible for a familial form of Parkinsons disease and may be involved in sporadic forms as well. Trempe et al. (p. 1451, published online 9 May) present the crystal structure of full-length parkin in an autoinhibited configuration. Guided by the structure, mutations were designed that activated parkin both in vitro and in cells. Because parkin is neuroprotective, the structure provides a framework for enhancing parkin function as a therapeutic strategy in Parkinsons disease. The complete structure of a protein linked to Parkinson’s disease suggests how to activate it. Mutations in the PARK2 (parkin) gene are responsible for an autosomal recessive form of Parkinson’s disease. The parkin protein is a RING-in-between-RING E3 ubiquitin ligase that exhibits low basal activity. We describe the crystal structure of full-length rat parkin. The structure shows parkin in an autoinhibited state and provides insight into how it is activated. RING0 occludes the ubiquitin acceptor site Cys431 in RING2, whereas a repressor element of parkin binds RING1 and blocks its E2-binding site. Mutations that disrupted these inhibitory interactions activated parkin both in vitro and in cells. Parkin is neuroprotective, and these findings may provide a structural and mechanistic framework for enhancing parkin activity.

An NMR approach to structural proteomics

The influx of genomic sequence information has led to the concept of structural proteomics, the determination of protein structures on a genome-wide scale. Here we describe an approach to structural proteomics of small proteins using NMR spectroscopy. Over 500 small proteins from several organisms were cloned, expressed, purified, and evaluated by NMR. Although there was variability among proteomes, overall 20% of these proteins were found to be readily amenable to NMR structure determination. NMR sample preparation was centralized in one facility, and a distributive approach was used for NMR data collection and analysis. Twelve structures are reported here as part of this approach, which allowed us to infer putative functions for several conserved hypothetical proteins.

Structure and function of the C-terminal PABC domain of human poly(A)-binding protein

We have determined the solution structure of the C-terminal quarter of human poly(A)-binding protein (hPABP). The protein fragment contains a protein domain, PABC [for poly(A)-binding protein C-terminal domain], which is also found associated with the HECT family of ubiquitin ligases. By using peptides derived from PABP interacting protein (Paip) 1, Paip2, and eRF3, we show that PABC functions as a peptide binding domain. We use chemical shift perturbation analysis to identify the peptide binding site in PABC and the major elements involved in peptide recognition. From comparative sequence analysis of PABC-binding peptides, we formulate a preliminary PABC consensus sequence and identify human ataxin-2, the protein responsible for type 2 spinocerebellar ataxia (SCA2), as a potential PABC ligand.

A structural overview of the PDI family of proteins.

Protein disulfide isomerases (PDIs) are enzymes that mediate oxidative protein folding in the endoplasmic reticulum. Understanding of PDIs has historically been hampered by lack of structural information. Over the last several years, partial and full‐length PDI structures have been solved at an increasing rate. Analysis of the structures reveals common features shared by several of the best known PDI family members, and also unique features related to substrate and partner binding sites. These exciting breakthroughs provide a deeper understanding of the mechanisms of oxidative protein folding in cells.

Specific interaction of ERp57 and calnexin determined by NMR spectroscopy and an ER two‐hybrid system

Calnexin and ERp57 act cooperatively to ensure a proper folding of proteins in the endoplasmic reticulum (ER). Calnexin contains two domains: a lectin domain and an extended arm termed the P‐domain. ERp57 is a protein disulfide isomerase composed of four thioredoxin‐like repeats and a short basic C‐terminal tail. Here we show direct interactions between the tip of the calnexin P‐domain and the ERp57 basic C‐terminus by using NMR and a novel membrane yeast two‐hybrid system (MYTHS) for mapping protein interactions of ER proteins. Our results prove that a small peptide derived from the P‐domain is active in binding ERp57, and we determine the structure of the bound conformation of the P‐domain peptide. The experimental strategy of using the MYTHS two‐hybrid system to map interaction sites between ER proteins, together with NMR, provides a powerful new strategy for establishing the function of ER complexes.

SH3 domains from a subset of BAR proteins define a Ubl-binding domain and implicate parkin in synaptic ubiquitination.

Mutations in the parkin gene are responsible for a common inherited form of Parkinsons disease (PD). Parkin is a RING-type E3 ubiquitin ligase with an N-terminal ubiquitin-like domain (Ubl). We report here that the parkin Ubl binds SH3 domains from endocytic BAR proteins such as endophilin-A with an affinity comparable to proline-rich domains (PRDs) from well-established SH3 partners. The NMR structure of the Ubl-SH3 complex identifies the PaRK extension, a unique C-terminal motif in the parkin Ubl required for SH3 binding and for parkin-mediated ubiquitination of endophilin-A in vitro. In nerve terminals, conditions that promote phosphorylation enhance the interaction between parkin and endophilin-A and increase the levels of ubiquitinated proteins within PRD-associated synaptic protein complexes in wild-type but not parkin knockout brain. The findings identify a pathway for the recruitment of synaptic substrates to parkin with the potential to explain the defects in synaptic transmission observed in recessive forms of PD.

Structural basis of ligand recognition by PABC, a highly specific peptide-binding domain found in poly(A)-binding protein and a HECT ubiquitin ligase

The C‐terminal domain of poly(A)‐binding protein (PABC) is a peptide‐binding domain found in poly(A)‐binding proteins (PABPs) and a HECT (homologous to E6‐AP C‐terminus) family E3 ubiquitin ligase. In protein synthesis, the PABC domain of PABP functions to recruit several translation factors possessing the PABP‐interacting motif 2 (PAM2) to the mRNA poly(A) tail. We have determined the solution structure of the human PABC domain in complex with two peptides from PABP‐interacting protein‐1 (Paip1) and Paip2. The structures show a novel mode of peptide recognition, in which the peptide binds as a pair of β‐turns with extensive hydrophobic, electrostatic and aromatic stacking interactions. Mutagenesis of PABC and peptide residues was used to identify key protein–peptide interactions and quantified by isothermal calorimetry, surface plasmon resonance and GST pull‐down assays. The results provide insight into the specificity of PABC in mediating PABP–protein interactions.

Solution structure of the bb′ domains of human protein disulfide isomerase

Protein disulfide isomerase is the most abundant and best studied of the disulfide isomerases that catalyze disulfide bond formation in the endoplasmic reticulum, yet the specifics of how it binds substrate have been elusive. Protein disulfide isomerase is composed of four thioredoxin‐like domains (abb′a′). Cross‐linking studies with radiolabeled peptides and unfolded proteins have shown that it binds incompletely folded proteins primarily via its third domain, b′. Here, we determined the solution structure of the second and third domains of human protein disulfide isomerase (b and b′, respectively) by triple‐resonance NMR spectroscopy and molecular modeling. NMR titrations identified a large hydrophobic surface within the b′ domain that binds unfolded ribonuclease A and the peptides mastoparan and somatostatin. Protein disulfide isomerase‐catalyzed refolding of reduced ribonuclease A in vitro was inhibited by these peptides at concentrations equal to their affinity to the bb′ fragment. Our findings provide a structural basis for previous kinetic and cross‐linking studies which have shown that protein disulfide isomerase exhibits a saturable, substrate‐binding site.

Poly(A) binding protein (PABP) homeostasis is mediated by the stability of its inhibitor, Paip2

The poly(A)‐binding protein (PABP) is a unique translation initiation factor in that it binds to the mRNA 3′ poly(A) tail and stimulates recruitment of the ribosome to the mRNA at the 5′ end. PABP activity is tightly controlled by the PABP‐interacting protein 2 (Paip2), which inhibits translation by displacing PABP from the mRNA. Here, we describe a close interplay between PABP and Paip2 protein levels in the cell. We demonstrate a mechanism for this co‐regulation that involves an E3 ubiquitin ligase, EDD, which targets Paip2 for degradation. PABP depletion by RNA interference (RNAi) causes co‐depletion of Paip2 protein without affecting Paip2 mRNA levels. Upon PABP knockdown, Paip2 interacts with EDD, which leads to Paip2 ubiquitination. Supporting a critical role for EDD in Paip2 degradation, knockdown of EDD expression by siRNA leads to an increase in Paip2 protein stability. Thus, we demonstrate that the turnover of Paip2 in the cell is mediated by EDD and is regulated by PABP. This mechanism serves as a homeostatic feedback to control the activity of PABP in cells.