G Protein Coupled Receptors (GPCRs) are critically regulated by β-arrestins (βarrs), which not only desensitize G protein signaling but also initiate a G protein independent wave of signaling1-5. A recent surge of structural data on a number of GPCRs, including the β2 adrenergic receptor (β2AR)-G protein complex, has provided novel insights into the structural basis of receptor activation6-11. Lacking however has been complementary information on recruitment of βarrs to activated GPCRs primarily due to challenges in obtaining stable receptor-βarr complexes for structural studies. Here, we devised a strategy for forming and purifying a functional β2AR-βarr1 complex that allowed us to visualize its architecture by single particle negative stain electron microscopy (EM) and to characterize the interactions between β2AR and βarr1 using hydrogen-deuterium exchange mass spectrometry (HDXMS) and chemical cross-linking. EM 2D averages and 3D reconstructions reveal bimodal binding of βarr1 to the β2AR, involving two separate sets of interactions, one with the phosphorylated carboxy-terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of cross-linked residues suggest engagement of the finger loop of βarr1 with the seven-transmembrane core of the receptor. In contrast, focal areas of increased HDX indicate regions of increased dynamics in both N and C domains of βarr1 when coupled to the β2AR. A molecular model of the β2AR-βarr signaling complex was made by docking activated βarr1 and β2AR crystal structures into the EM map densities with constraints provided by HDXMS and cross-linking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented herein provides a framework for better understanding the basis of GPCR regulation by arrestins.

Mammalian centromeres are not defined by a consensus DNA sequence. In all eukaryotes a hallmark of functional centromeres--both normal ones and those formed aberrantly at atypical loci--is the accumulation of centromere protein A (CENP-A), a histone variant that replaces H3 in centromeric nucleosomes. Here we show using deuterium exchange/mass spectrometry coupled with hydrodynamic measures that CENP-A and histone H4 form sub-nucleosomal tetramers that are more compact and conformationally more rigid than the corresponding tetramers of histones H3 and H4. Substitution into histone H3 of the domain of CENP-A responsible for compaction is sufficient to direct it to centromeres. Thus, the centromere-targeting domain of CENP-A confers a unique structural rigidity to the nucleosomes into which it assembles, and is likely to have a role in maintaining centromere identity.

Summary G protein-coupled receptors (GPCRs) represent the largest family of membrane receptors1 that instigate signaling through nucleotide exchange on heterotrimeric G proteins. Nucleotide exchange, or more precisely GDP dissociation from the G protein α-subunit, is the key step toward G protein activation and initiation of downstream signaling cascades. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G protein activation remain elusive. To characterize this mechanism we applied peptide amide hydrogen-deuterium exchange mass spectrometry (DXMS) to probe changes in the structure of the heterotrimeric G protein Gs (the stimulatory G protein for adenylyl cyclase) upon formation of a complex with agonist-bound β2 adrenergic receptor (β2AR). Our studies reveal structural links between the receptor binding surface and the nucleotide-binding pocket of Gs that undergo higher levels of hydrogen-deuterium exchange (HX) than would be predicted from the crystal structure of the β2AR-Gs complex. Together with x-ray crystallographic and electron microscopic data of the β2AR-Gs complex (ref 2 and Westfield et al, manuscript submitted), we provide a rationale for a mechanism of nucleotide exchange whereby the receptor perturbs the structure of the amino-terminal region of α-subunit of Gs and consequently alters the ‘P-loop’ that binds the β-phosphate in GDP. As with the ras-family of small molecular weight G proteins, P-loop stabilization and β-phosphate coordination are key determinants of GDP (and GTP) binding affinity.

Understanding the energetics of molecular interactions is fundamental to all of the central quests of structural biology including structure prediction and design, mapping evolutionary pathways, learning how mutations cause disease, drug design, and relating structure to function. Hydrogen-bonding is widely regarded as an important force in a membrane environment because of the low dielectric constant of membranes and a lack of competition from water. Indeed, polar residue substitutions are the most common disease-causing mutations in membrane proteins. Because of limited structural information and technical challenges, however, there have been few quantitative tests of hydrogen-bond strength in the context of large membrane proteins. Here we show, by using a double-mutant cycle analysis, that the average contribution of eight interhelical side-chain hydrogen-bonding interactions throughout bacteriorhodopsin is only 0.6 kcal mol(-1). In agreement with these experiments, we find that 4% of polar atoms in the non-polar core regions of membrane proteins have no hydrogen-bond partner and the lengths of buried hydrogen bonds in soluble proteins and membrane protein transmembrane regions are statistically identical. Our results indicate that most hydrogen-bond interactions in membrane proteins are only modestly stabilizing. Weak hydrogen-bonding should be reflected in considerations of membrane protein folding, dynamics, design, evolution and function.

The active-state complex between an agonist-bound receptor and a guanine nucleotide-free G protein represents the fundamental signaling assembly for the majority of hormone and neurotransmitter signaling. We applied single-particle electron microscopy (EM) analysis to examine the architecture of agonist-occupied β 2 -adrenoceptor (β 2 AR) in complex with the heterotrimeric G protein Gs (Gαsβγ). EM 2D averages and 3D reconstructions of the detergent-solubilized complex reveal an overall architecture that is in very good agreement with the crystal structure of the active-state ternary complex. Strikingly however, the α-helical domain of Gαs appears highly flexible in the absence of nucleotide. In contrast, the presence of the pyrophosphate mimic foscarnet (phosphonoformate), and also the presence of GDP, favor the stabilization of the α-helical domain on the Ras-like domain of Gαs. Molecular modeling of the α-helical domain in the 3D EM maps suggests that in its stabilized form it assumes a conformation reminiscent to the one observed in the crystal structure of Gαs-GTPγS. These data argue that the α-helical domain undergoes a nucleotidedependent transition from a flexible to a conformationally stabilized state.G protein-coupled receptor | negative stain electron microscopy | random conical tilt T he majority of hormones and neurotransmitters communicate information to cells via G protein-coupled receptors (GPCRs), which instigate intracellular signaling by activating their cognate heterotrimeric G proteins on the cytoplasmic side. GPCRs constitute the largest family of membrane proteins and play essential roles in regulating every aspect of normal physiology, thereby representing major pharmacological targets. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G-protein activation remain elusive. The β 2 -adrenergic receptor (β 2 AR) and its complex with heterotrimeric stimulatory G-protein Gs (Gαsβγ) represent an ideal model system for the large family of GPCRs activated by diffusible ligands. Agonist binding to the β 2 AR promotes interactions with GDP-bound Gsαβγ heterotrimer, leading to the exchange of GDP for GTP, and the functional dissociation of Gs into Gα-GTP and Gβγ subunits. To examine the architecture of agonist occupied β 2 AR in complex with Gαsβγ under different conditions, we used electron microscopy (EM) and single-particle analysis. Because of the limited size of the protein complex (∼148 kDa), we visualized specimens embedded in negative stain, which provides sufficient contrast from relatively small protein assemblies (1). This approach allowed us to obtain 2D projection averages and 3D reconstructions that provided new insights into dynamic features of the β 2 AR-Gs complex, and helped guide a successful approach to crystallize the complex enabling a high-resolution structure (2). Results and DiscussionIn a first step, we sought to examine the architecture of complexes in the nucleot...

The structure of ␣-synuclein (␣-syn) amyloid was studied by hydrogen-deuterium exchange by using a fragment separation-MS analysis. The conditions used made it possible to distinguish the exchange of unprotected and protected amide hydrogens and to define the order͞disorder boundaries at close to amino acid resolution. The soluble ␣-syn monomer exchanges its amide hydrogens with water hydrogens at random coil rates, consistent with its natively unstructured condition. In assembled amyloid, long N-terminal and C-terminal segments remain unprotected (residues 1-Ϸ38 and 102-140), although the N-terminal segment shows some heterogeneity. A continuous middle segment (residues Ϸ39 -101) is strongly protected by systematically H-bonded cross-␤ structure. This segment is much too long to fit the amyloid ribbon width, but non-H-bonded amides expected for directionchanging loops are not apparent. These results and other known constraints specify that ␣-syn amyloid adopts a chain fold like that suggested before for amyloid-␤ [Petkova et al. M any proteins and polypeptides are able to adopt the generic massively aggregated structure known as amyloid (1). The macroscopic fibrillar character of amyloid is obvious by direct electron microscopic observation, but its detailed structure and the structural basis of its unusual behavior remains a challenging problem (2-9).Methods based on the hydrogen exchange (HX) behavior of polypeptides can provide useful information. The backbone amide hydrogens of proteins engage in continual exchange with the hydrogens of solvent water. These hydrogens, uniformly distributed at every amino acid (except proline) in every protein molecule, provide built-in, structure-sensitive, nonperturbing probes that can be used to study soluble or insoluble proteins under any desired conditions. Hydrogens that are freely exposed to solvent exchange at known rates that depend on pH, temperature, neighboring residues, and the hydrogen isotopes used (10,11). Hydrogens that are protected by structure, almost always in H bonds, exchange far more slowly. Their exchange is modulated by dynamic structural events that reversibly separate protecting H bonds and transiently expose them to the normal chemical exchange process. Accordingly, HX measurements can distinguish the presence and absence of protecting structure, determine the thermodynamic stability and dynamic properties of local and surrounding structure, and probe the effects of mutations, manipulations, and conditions, in principle, at amino acid resolution (12).The development of multidimensional solution NMR methods (13-15) has made high-resolution analysis of HX behavior routine for small soluble proteins (16). A fragment separation method that does not depend on solution NMR measurement extends HX studies to large proteins and insoluble protein systems (17)(18)(19). In this method, hydrogen isotope exchange can be performed under conditions that are most pertinent for the protein system being studied. Timed samples are then placed into slow HX condition...

An automated high-throughput, high-resolution deuterium exchange HPLC-MS method (DXMS) was used to extend previous hydrogen exchange studies on the position and energetic role of regulatory structure changes in hemoglobin. The results match earlier highly accurate but much more limited tritium exchange results, extend the analysis to the entire sequence of both hemoglobin subunits, and identify some energetically important changes. Allosterically sensitive amide hydrogens located at near amino acid resolution help to confirm the reality of local unfolding reactions and their use to evaluate resolved structure changes in terms of allosteric free energy.H ydrogen exchange (HX) measurements can, in principle, locate protein-binding sites and structure changes and can quantify otherwise unavailable dynamic and energetic parameters (1-4). For relatively small proteins, HX can be measured at an amino acid resolved level by NMR methods. For larger, functionally more interesting proteins, other strategies are required. Earlier work (5, 6) developed a ''functional-labeling'' approach that can selectively label, by hydrogen-tritium (H-T) or hydrogen-deuterium (H-D) exchange, just those sites that change in any functional process. In favorable cases, the label can then be located at medium resolution by a proteolytic fragmentation method in which the fragments are quickly produced and then separated by HPLC under conditions where the loss of isotopic label is slow (6-9).To move toward higher resolution and more comprehensive coverage of target proteins, recent work in many laboratories has coupled the HPLC separation to a second dimension of fragment resolution by online MS (10, 11). These methods tend to be labor intensive and time consuming, with limitations in throughput and comprehensiveness and in the structural resolution of functionally important changes. This article merges previous HX functional labeling and fragment separation methods with an automated MS approach termed deuterium exchange MS (DXMS) (12-18).We are using Hb as a model system to study how protein molecules manage intramolecular signal transduction processes. Hb functions by transducing a part of the binding energy of its initially bound O 2 ligands into structure-change energy. The energy is carried through the protein to distant heme sites in the form of energetic structure changes, and there transduced back into binding energy. The initial reduced binding energy and the later enhanced binding produces the physiologically important sigmoid binding curve. In short, the currency of allosteric interactions is free energy. Trying to understand allostery without measuring free energy is like trying to understand an economic system without measuring money. A great deal of information on regulatory structure change in many proteins is now available, but mainly in a qualitative pictorial sense from ''before and after'' crystallographic or NMR views. How these changes participate in energy transduction and translocation has been little explored (19)(20)(21...

Arenaviruses cause disease in industrialized and developing nations alike. Among them, the hemorrhagic fever virus Lassa is responsible for ∼300,000-500,000 infections/y in Western Africa. The arenavirus nucleoprotein (NP) forms the protein scaffold of the genomic ribonucleoprotein complexes and is critical for transcription and replication of the viral genome. Here, we present crystal structures of the RNA-binding domain of Lassa virus NP in complex with ssRNA. This structure shows, in contrast to the predicted model, that RNA binds in a deep, basic crevice located entirely within the N-terminal domain. Furthermore, the NP-ssRNA structures presented here, combined with hydrogen-deuterium exchange/MS and functional studies, suggest a gating mechanism by which NP opens to accept RNA. Directed mutagenesis and functional studies provide a unique look into how the arenavirus NPs bind to and protect the viral genome and also suggest the likely assembly by which viral ribonucleoprotein complexes are organized.structural biology | virology T he arenavirus family has a worldwide distribution and contains significant human pathogens such as Lassa (LASV), Machupo, Junin, Lujo (1, 2), and lymphyocytic choriomeningitis virus. Of these arenaviruses, LASV carries the largest disease burden, causing 300,000 to 500,000 infections per year in Western Africa. It is also the hemorrhagic fever most frequently transported out of Africa to the United States and Europe (2-4).Arenaviruses have a bisegmented, negative-sense, singlestranded RNA genome with a unique ambisense coding strategy that produces just four known proteins: a glycoprotein, a nucleoprotein (NP), a matrix protein (Z), and a polymerase (L) (2). Of these proteins, NP is the most abundant in an infected cell. NP associates with L to form the ribonucleoprotein (RNP) core for RNA replication and transcription (5) and the matrix protein Z for viral assembly (6-8). The arenavirus NP also plays an important role in the suppression of the innate immune system (9-11).Genome and antigenome RNAs of negative-strand RNA viruses (NSV) do not exist as naked RNA, but rather as a RNP complex in which the RNA is encapsidated by the viral nucleoprotein. During replication of many negative-strand RNA viruses, the nascent nucleoprotein (usually termed N) is bound by a polymerase cofactor (often a phosphoprotein, termed P), which prevents polymerization of N and nonspecific encapsidation of host cell RNAs (12-15). The resulting complex is termed N 0 -P, in which N 0 denotes RNA-free N. The arenavirus, orthomyxovirus (flu), and bunyavirus (Hanta, Rift Valley Fever) families (i.e., segmented NSV) do not encode an analogous P protein, and the mechanism by which the nucleoprotein controls RNA binding during virus infection is not yet understood.The arenavirus nucleoprotein (termed NP instead of N) has distinct N-and C-terminal domains connected by a flexible linker (16)(17)(18)(19). The C-terminal domain functions as an exonuclease (16, 17) specific for dsRNA (17) and linked to antagonism of t...