Yulia Pustovalova

Spatial Structure of the Transmembrane Domain Heterodimer of ErbB1 and ErbB2 Receptor Tyrosine Kinases

Growth factor receptor tyrosine kinases of the ErbB family play a significant role in vital cellular processes and various cancers. During signal transduction across plasma membrane, ErbB receptors are involved in lateral homodimerization and heterodimerization with proper assembly of their extracellular single-span transmembrane (TM) and cytoplasmic domains. The ErbB1/ErbB2 heterodimer appears to be the strongest and most potent inducer of cellular transformation and mitogenic signaling compared to other ErbB homodimers and heterodimers. Spatial structure of the heterodimeric complex formed by TM domains of ErbB1 and ErbB2 receptors embedded into lipid bicelles was obtained by solution NMR. The ErbB1 and ErbB2 TM domains associate in a right-handed alpha-helical bundle through their N-terminal double GG4-like motif T(648)G(649)X(2)G(652)A(653) and glycine zipper motif T(652)X(3)S(656)X(3)G(660), respectively. The described heterodimer conformation is believed to support the juxtamembrane and kinase domain configuration corresponding to the receptor active state. The capability for multiple polar interactions, along with hydrogen bonding between TM segments, correlates with the observed highest affinity of the ErbB1/ErbB2 heterodimer, implying an important contribution of the TM helix-helix interaction to signal transduction.

Alternative packing of EGFR transmembrane domain suggests that protein–lipid interactions underlie signal conduction across membrane

The human epidermal growth factor receptor (EGFR) of HER/ErbB receptor tyrosine kinase family mediates a broad spectrum of cellular responses transducing biochemical signals via lateral dimerization in plasma membrane, while inactive receptors can exist in both monomeric and dimeric forms. Recently, the dimeric conformation of the helical single-span transmembrane domains of HER/ErbB employing the relatively polar N-terminal motifs in a fashion permitting proper kinase activation was experimentally determined. Here we describe the EGFR transmembrane domain dimerization via an alternative weakly polar C-terminal motif A(661)xxxG(665) presumably corresponding to the inactive receptor state. During association, the EGFR transmembrane helices undergo a structural adjustment with adaptation of inter-molecular polar and hydrophobic interactions depending upon the surrounding membrane properties that directly affect the transmembrane helix packing. This might imply that signal transduction through membrane and allosteric regulation are inclusively mediated by coupled protein-protein and protein-lipid interactions, elucidating paradoxically loose linkage between ligand binding and kinase activation.

NMR Mapping of PCNA Interaction with Translesion Synthesis DNA Polymerase Rev1 Mediated by Rev1-BRCT Domain

Rev1 is a Y-family translesion synthesis (TLS) DNA polymerase involved in bypass replication across sites of DNA damage and postreplicational gap filling. In the process of TLS, high-fidelity replicative DNA polymerases stalled by DNA damage are replaced by error-prone TLS enzymes responsible for the majority of mutagenesis in eukaryotic cells. The polymerase exchange that gains low-fidelity TLS polymerases access to DNA is mediated by their interactions with proliferating cell nuclear antigen (PCNA). Rev1 stands alone from other Y-family TLS enzymes since it lacks the consensus PCNA-interacting protein box (PIP-box) motif, instead utilizing other modular domains for PCNA binding. Here we report solution NMR structure of an 11-kDa BRCA1 C-terminus (BRCT) domain from Saccharomyces cerevisiae Rev1 and demonstrate with the use of transverse relaxation optimized spectroscopy (TROSY) NMR methods that Rev1-BRCT domain directly interacts with an 87-kDa PCNA in solution. The domain adopts α/β fold (β1-α1-β2-β3-α2-β4-α3-α4) typical for BRCT domain superfamily. PCNA-binding interface of the Rev1-BRCT domain comprises conserved residues of the outer surface of the α1-helix and the α1-β1, β2-β3 and β3-α2 loops. On the other hand, Rev1-BRCT binds to the inter-domain region of PCNA that overlaps with the binding site for the PIP-box motif. Furthermore, Rev1-BRCT domain bound to PCNA can be displaced by increasing amounts of the PIP-box peptide from TLS DNA polymerase polη, suggesting that Rev1-BRCT and polη PIP-box interactions with the same PCNA monomer are mutually exclusive. These results provide structural insights into PCNA recognition by TLS DNA polymerases that help better understand TLS regulation in eukaryotes.

The C-terminal domain of human Rev1 contains independent binding sites for DNA polymerase η and Rev7 subunit of polymerase ζ.

REV1 , REV3 , REV7 and Polη‐RIR peptide physically interact by nuclear magnetic resonance (View interaction).

PHD domain from human SHPRH

SHPRH (SNF2, histone linker, PHD, RING, helicase) is a SWI2/SNF2-family ATP-dependent chromatin remodeling factor, and one of E3 ubiquitin ligases responsible for Ubc13-Mms2-dependent K63 poly-ubiquitination of PCNA (proliferating cell nuclear antigen) that promotes error-free DNA damage tolerance in eukaryotes. In contrast to its functional homologues, S. cerevisiae Rad5 and human HLTF (helicase like transcription factor), SHPRH contains a PHD (plant homeodomain) finger embedded in the ‘minor’ insert region of the core helicase-like domain. PHD fingers are often found in proteins involved in chromatin remodeling and transcription regulation, and are generally considered as ‘readers’ of methylation state of histone tails, primarily the lysine 4 (K4) residue of histone H3 (H3K4). Here we report the solution NMR structure of the SHPRH PHD domain and investigate whether this domain is capable of recognizing H3K4 modifications. The domain adopts a canonical PHD-finger fold with a central two-stranded anti-parallel β-sheet flanked on both sides by the two interleaved zinc-binding sites. Despite the presence of a subset of aromatic residues characteristic for PHD-fingers that preferentially bind methylated H3K4, NMR titration experiments reveal that SHPRH PHD does not specifically interact with the H3-derived peptides irrespective of K4 methylation. This result suggests that the SHPRH PHD domain might have evolved a different function other than recognizing histone modifications.

Structural Characterization of the Early Events in the Nucleation–Condensation Mechanism in a Protein Folding Process

The nucleation-condensation mechanism represents a major paradigm to understand the folding process of many small globular proteins. Although substantial evidence has been acquired for this mechanism, it has remained very challenging to characterize the initial events leading to the formation of a folding nucleus. To achieve this goal, we used a combination of relaxation dispersion NMR spectroscopy and molecular dynamics simulations to determine ensembles of conformations corresponding to the denatured, transition, and native states in the folding of the activation domain of human procarboxypeptidase A2 (ADA2h). We found that the residues making up the folding nucleus tend to interact in the denatured state in a transient manner and not simultaneously, thereby forming incomplete and distorted versions of the folding nucleus. Only when all the contacts between these key residues are eventually formed can the protein reach the transition state and continue folding. Overall, our results elucidate the mechanism of formation of the folding nucleus of a protein and provide insights into how its folding rate can be modified during evolution by mutations that modulate the strength of the interactions between the residues forming the folding nucleus.

Impact of membrane partitioning on the spatial structure of an S-type cobra cytotoxin.

Cobra cytotoxins (CTs) belong to the three-fingered protein family. They are classified into S- and P-types, the latter exhibiting higher membrane-perturbing capacity. In this work, we investigated the interaction of CTs with phospholipid bilayers, using coarse-grained (CG) and full-atom (FA) molecular dynamics (MD). The object of this work is a CT of an S-type, cytotoxin I (CT1) from N.oxiana venom. Its spatial structure in aqueous solution and in the micelles of dodecylphosphocholine (DPC) were determined by 1H-NMR spectroscopy. Then, via CG- and FA MD-computations, we evaluated partitioning of CT1 molecule into palmitoyloleoylphosphatidylcholine (POPC) membrane, using the toxin spatial models, obtained either in aqueous solution, or detergent micelle. The latter model exhibits minimal structural changes upon partitioning into the membrane, while the former deviates from the starting conformation, loosing the tightly bound water molecule in the loop-2. These data show that the structural changes elicited by CT1 molecule upon incorporation into DPC micelle take place likely in the lipid membrane, although the mode of the interaction of this toxin with DPC micelle (with the tips of the all three loops) is different from its mode in POPC membrane (primarily with the tip of the loop-1 and both the tips of the loop-1 and loop-2).

Probing the Residual Structure of the Low Populated Denatured State of ADA2h under Folding Conditions by Relaxation Dispersion Nuclear Magnetic Resonance Spectroscopy

The structural characterization of low populated states of proteins with accuracy comparable to that achievable for native states is important for understanding the mechanisms of protein folding and function, as well as misfolding and aggregation. Because of the transient nature of these low populated states, they are seldom detected directly under conditions that favor folding. The activation domain of human procarboxypeptidase A2 (ADA2h) is an α/β-protein that forms amyloid fibrils at low pH, presumably initiated from a denatured state with a considerable amount of residual structure. Here we used Carr-Parcell-Meiboom-Gill relaxation dispersion (CPMG RD) nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the denatured state of the ADA2h I71V mutant under conditions that favor folding. Under these conditions, the lifetime of the denatured state of I71V ADA2h is on the order of milliseconds and its population is approximately several percent, which makes this mutant amenable to studies by CPMG RD methods. The nearly complete set of CPMG RD-derived backbone (15)N, (13)C, and (1)H NMR chemical shifts in the I71V ADA2h denatured state reveals that it retains a significant fraction (up to 50-60%) of nativelike α-helical structure, while the regions encompassing native β-strands are structured to a much lesser extent. The nativelike α-helical structure of the denatured state can bring together hydrophobic residues on the same sides of α-helices, making them available for intra- or intermolecular interactions. CPMG RD data analysis thus allowed a detailed structural characterization of the ADA2h denatured state under folding conditions not previously achieved for this protein.