Brian O. Smith

The structure of mouse HP1 suggests a unique mode of single peptide recognition by the shadow chromo domain dimer

The heterochromatin protein 1 (HP1) family of proteins is involved in gene silencing via the formation of heterochromatic structures. They are composed of two related domains: an N‐terminal chromo domain and a C‐terminal shadow chromo domain. Present results suggest that chromo domains may function as protein interaction motifs, bringing together different proteins in multi‐protein complexes and locating them in heterochromatin. We have previously determined the structure of the chromo domain from the mouse HP1β protein, MOD1. We show here that, in contrast to the chromo domain, the shadow chromo domain is a homodimer. The intact HP1β protein is also dimeric, where the interaction is mediated by the shadow chromo domain, with the chromo domains moving independently of each other at the end of flexible linkers. Mapping studies, with fragments of the CAF1 and TIF1β proteins, show that an intact, dimeric, shadow chromo domain structure is required for complex formation.

Plant UVR8 Photoreceptor Senses UV-B by Tryptophan-Mediated Disruption of Cross-Dimer Salt Bridges

Donuts Dissociate In Arabidopsis, the UVR8 protein responds to ultraviolet-B (UV-B) light by dissociating into monomers, which are then available to interact with downstream factors that enact the plants response to light. Christie et al. (p. 1492, published online 9 February; see the cover and see the Perspective by Gardner and Correa) have now determined the crystal structure of UVR8. Without ultraviolet-B light, UVR8 dimerizes, with two donut-shaped monomers joined by a network of salt bridges. Close-packing of a pyramid of tryptophan residues permits exciton coupling that is key to UV-B perception. Electron transfer after UV-B perception could dissociate the salt bridges that hold the dimer together and release monomeric UVR8 to initiate light-induced signaling. A tryptophan pyramid allows a dimeric protein to perceive ultraviolet light without an additional chromophore. The recently identified plant photoreceptor UVR8 (UV RESISTANCE LOCUS 8) triggers regulatory changes in gene expression in response to ultraviolet-B (UV-B) light through an unknown mechanism. Here, crystallographic and solution structures of the UVR8 homodimer, together with mutagenesis and far-UV circular dichroism spectroscopy, reveal its mechanisms for UV-B perception and signal transduction. β-propeller subunits form a remarkable, tryptophan-dominated, dimer interface stitched together by a complex salt-bridge network. Salt-bridging arginines flank the excitonically coupled cross-dimer tryptophan “pyramid” responsible for UV-B sensing. Photoreception reversibly disrupts salt bridges, triggering dimer dissociation and signal initiation. Mutation of a single tryptophan to phenylalanine retunes the photoreceptor to detect UV-C wavelengths. Our analyses establish how UVR8 functions as a photoreceptor without a prosthetic chromophore to promote plant development and survival in sunlight.

Protein structure determination in living cells by in-cell NMR spectroscopy

Investigating proteins ‘at work’ in a living environment at atomic resolution is a major goal of molecular biology, which has not been achieved even though methods for the three-dimensional (3D) structure determination of purified proteins in single crystals or in solution are widely used. Recent developments in NMR hardware and methodology have enabled the measurement of high-resolution heteronuclear multi-dimensional NMR spectra of macromolecules in living cells (in-cell NMR). Various intracellular events such as conformational changes, dynamics and binding events have been investigated by this method. However, the low sensitivity and the short lifetime of the samples have so far prevented the acquisition of sufficient structural information to determine protein structures by in-cell NMR. Here we show the first, to our knowledge, 3D protein structure calculated exclusively on the basis of information obtained in living cells. The structure of the putative heavy-metal binding protein TTHA1718 from Thermus thermophilus HB8 overexpressed in Escherichia coli cells was solved by in-cell NMR. Rapid measurement of the 3D NMR spectra by nonlinear sampling of the indirectly acquired dimensions was used to overcome problems caused by the instability and low sensitivity of living E. coli samples. Almost all of the expected backbone NMR resonances and most of the side-chain NMR resonances were observed and assigned, enabling high quality (0.96 ångström backbone root mean squared deviation) structures to be calculated that are very similar to the in vitro structure of TTHA1718 determined independently. The in-cell NMR approach can thus provide accurate high-resolution structures of proteins in living environments.

Crystal structure of the complex of the cyclin D-dependent kinase Cdk6 bound to the cell-cycle inhibitor p19INK4d.

The crystal structure of the cyclin D-dependent kinase Cdk6 bound to the p19INK4d protein has been determined at 1.9 Å resolution. The results provide the first structural information for a cyclin D-dependent protein kinase and show how the INK4 family of CDK inhibitors bind. The structure indicates that the conformational changes induced by p19INK4d inhibit both productive binding of ATP and the cyclin-induced rearrangement of the kinase from an inactive to an active conformation. The structure also shows how binding of an INK4 inhibitor would prevent binding of p27Kip1, resulting in its redistribution to other CDKs. Identification of the critical residues involved in the interaction explains how mutations in Cdk4 and p16INK4a result in loss of kinase inhibition and cancer.

EPAC proteins transduce diverse cellular actions of cAMP

It has now been over 10 years since efforts to completely understand the signalling actions of cAMP (3′‐5′‐cyclic adenosine monophosphate) led to the discovery of exchange protein directly activated by cAMP (EPAC) proteins. In the current review we will highlight important advances in the understanding of EPAC structure and function and demonstrate that EPAC proteins mediate multiple actions of cAMP in cells, revealing future targets for pharmaceutical intervention. It has been known for some time that drugs that elevate intracellular cAMP levels have proven therapeutic benefit for diseases ranging from depression to inflammation. The challenge now is to determine which of these positive actions of cAMP involve activation of EPAC‐regulated signal transduction pathways. EPACs are specific guanine nucleotide exchange factors for the Ras GTPase homologues, Rap1 and Rap2, which they activate independently of the classical routes for cAMP signalling, cyclic nucleotide‐gated ion channels and protein kinase A. Rather, EPAC activation is triggered by internal conformational changes induced by direct interaction with cAMP. Leading from this has been the development of EPAC‐specific agonists, which has helped to delineate numerous cellular actions of cAMP that rely on subsequent activation of EPAC. These include regulation of exocytosis and the control of cell adhesion, growth, division and differentiation. Recent work also implicates EPAC in the regulation of anti‐inflammatory signalling in the vascular endothelium, namely negative regulation of pro‐inflammatory cytokine signalling and positive support of barrier function. Further elucidation of these important signalling mechanisms will no doubt support the development of the next generation of anti‐inflammatory drugs.

C-terminal region of the UV-B photoreceptor UVR8 initiates signaling through interaction with the COP1 protein

UV-B light initiates photomorphogenic responses in plants. Arabidopsis UV RESISTANCE LOCUS8 (UVR8) specifically mediates these responses by functioning as a UV-B photoreceptor. UV-B exposure converts UVR8 from a dimer to a monomer, stimulates the rapid accumulation of UVR8 in the nucleus, where it binds to chromatin, and induces interaction of UVR8 with CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), which functions with UVR8 to control photomorphogenic UV-B responses. Although the crystal structure of UVR8 reveals the basis of photoreception, it does not show how UVR8 initiates signaling through interaction with COP1. Here we report that a region of 27 amino acids from the C terminus of UVR8 (C27) mediates the interaction with COP1. The C27 region is necessary for UVR8 function in the regulation of gene expression and hypocotyl growth suppression in Arabidopsis. However, UVR8 lacking C27 still undergoes UV-B–induced monomerization in both yeast and plant protein extracts, accumulates in the nucleus in response to UV-B, and interacts with chromatin at the UVR8-regulated ELONGATED HYPOCOTYL5 (HY5) gene. The UV-B–dependent interaction of UVR8 and COP1 is reproduced in yeast cells and we show that C27 is both necessary and sufficient for the interaction of UVR8 with the WD40 domain of COP1. Furthermore, we show that C27 interacts in yeast with the REPRESSOR OF UV-B PHOTOMORPHOGENESIS proteins, RUP1 and RUP2, which are negative regulators of UVR8 function. Hence the C27 region has a key role in UVR8 function.

Structure of the cyclin-dependent kinase inhibitor p19Ink4d

In cancer, the biochemical pathways that are dominated by the two tumour-suppressor proteins, p53 and Rb, are the most frequently disrupted. Cyclin D-dependent kinases phosphorylate Rb to control its activity and they are, in turn, specifically inhibited by the Ink4 family of cyclin-dependent kinase inhibitors (CDKIs) which cause arrest at the G1 phase of the cell cycle. Mutations in Rb, cyclin D1, its catalytic subunit Cdk4, and the CDKI p16Ink4a, which alter the protein or its level of expression, are all strongly implicated in cancer. This suggests that the Rb ‘pathway’ is of particular importance. Here we report the structure of the p19Ink4d protein, determined by NMR spectroscopy. The structure indicates that most mutations to the p16Ink4a gene, which result in loss of function, are due to incorrectly folded and/or insoluble protein. We propose a model for the interaction of Ink4 proteins with D-type cyclin-Cdk4/6 complexes that might provide a basis for the design of therapeutics against cancer.

Structure of the C3b Binding Site of CR1 (CD35), the Immune Adherence Receptor

Complement receptor type 1 (CR1 or CD35) is a multiple modular protein that mediates the immune adherence phenomenon, a fundamental event for destroying microbes and initiating an immunological response. It fulfills this role through binding C3b/C4b-opsonized foreign antigens. The structure of the principal C3b/C4b binding site (residues 901-1095) of CR1 is reported, revealing three complement control protein modules (modules 15-17) in an extended head-to-tail arrangement with flexibility at the 16-17 junction. Structure-guided mutagenesis identified a positively charged surface region on module 15 that is critical for C4b binding. This patch, together with basic side chains of module 16 exposed on the same face of CR1, is required for C3b binding. These studies reveal the initial structural details of one of the first receptor-ligand interactions to be identified in immunobiology.

Identification of bacterial target proteins for the salicylidene acylhydrazide class of virulence blocking compounds

A class of anti-virulence compounds, the salicylidene acylhydrazides, has been widely reported to block the function of the type three secretion system of several Gram-negative pathogens by a previously unknown mechanism. In this work we provide the first identification of bacterial proteins that are targeted by this group of compounds. We provide evidence that their mode of action is likely to result from a synergistic effect arising from a perturbation of the function of several conserved proteins. We also examine the contribution of selected target proteins to the pathogenicity of Yersinia pseudotuberculosis and to expression of virulence genes in Escherichia coli O157.

Solution structure of the fibrin binding finger domain of tissue-type plasminogen activator determined by 1H nuclear magnetic resonance.

The amino acid sequence of the first domain of tissue-type plasminogen activator (t-PA) includes eight residues that are highly conserved in the type 1 finger domains found in human fibronectin. A construct comprising 50 residues from this finger domain of t-PA has been expressed and its solution structure has been determined by two-dimensional nuclear magnetic resonance spectroscopy. A total of 782 experimental restraints consisting of 723 interproton distances derived from nuclear Overhauser effect measurements, 43 torsion angles, and 16 hydrogen bond restraints were used as the input for dynamical simulated annealing structure calculations. Twenty-eight structures were obtained that satisfied the experimental data with no single distance violation greater than 0.3 A. The average atomic root-mean-square distribution for the backbone atoms of the final structures was 0.41 (+/- 0.13) A for the well defined part of the structure (residues 4 to 47). The overall fold of the t-PA finger domain shows a striking similarity to that of the seventh type 1 repeat of human fibronectin with the side-chains of conserved residues lying in similar conformations. One significant difference between the two molecules is that hydrophobic residues cover the exposed surface of the principal beta-sheet region in the t-PA finger domain. It is suggested that one face of this region may interact with parts of the complete t-PA protein.