Estimation of interdomain flexibility of N-terminus of factor H using residual dipolar couplings

Characterization of segmental flexibility is needed to understand the biological mechanisms of the very large category of functionally diverse proteins, exemplified by the regulators of complement activation, that consist of numerous compact modules or domains linked by short, potentially flexible, sequences of amino acid residues. The use of NMR-derived residual dipolar couplings (RDCs), in magnetically aligned media, to evaluate interdomain motion is established but only for two-domain proteins. We focused on the three N-terminal domains (called CCPs or SCRs) of the important complement regulator, human factor H (i.e. FH1-3). These domains cooperate to facilitate cleavage of the key complement activation-specific protein fragment, C3b, forming iC3b that no longer participates in the complement cascade. We refined a three-dimensional solution structure of recombinant FH1-3 based on nuclear Overhauser effects and RDCs. We then employed a rudimentary series of RDC datasets, collected in media containing magnetically aligned bicelles (disk-like particles formed from phospholipids) under three different conditions, to estimate interdomain motions. This circumvents a requirement of previous approaches for technically difficult collection of five independent RDC datasets. More than 80% of conformers of this predominantly extended three-domain molecule exhibit flexions of < 40 °. Such segmental flexibility (together with the local dynamics of the hypervariable loop within domain 3), could facilitate recognition of C3b via initial anchoring and eventual reorganization of modules to the conformation captured in the previously solved crystal structure of a C3b:FH1-4 complex

Protein-Protein Docking Using Long Range Nuclear Magnetic Resonance Constraints

One of the main methods for experimentally determining protein structure is nuclear magnetic resonance (NMR) spectroscopy. The advantage of using NMR compared to other methods is that the molecule may be studied in its natural state and environment. However, NMR is limited in its facility to analyze multi-domain molecules because of the scarcity of inter-atomic NMR constraints between the domains. In those cases it might be possible to dock the domains based on long range NMR constraints that are related to the molecule's overall structure. We present two computational methods for rigid docking based on long range NMR constraints. The first docking method is based on the overall alignment tensor of the complex. The docking algorithm is based on the minimization of the difference between the predicted and experimental alignment tensor. In order to efficiently dock the complex we introduce a new, computationally efficient method called PATI for predicting the molecular alignment tensor based on the three-dimensional structure of the molecule. The increase in speed compared to the currently best-known method (PALES) is achieved by re-expressing the problem as one of numerical integration, rather than a simple uniform sampling (as in the PALES method), and by using a convex hull rather than a detailed representation of the surface of a molecule. Using PATI, we derive a method called PATIDOCK for efficiently docking a two-domain complex based solely on the novel idea of using the difference between the experimental alignment tensor and the predicted alignment tensor computed by PATI. We show that the alignment tensor fundamentally contains enough information to accurately dock a two-domain complex, and that we can very quickly dock the two domains by pre-computing the right set of data. A second new docking method is based on a similar concept but using the rotational diffusion tensor. We derive a minimization algorithm for this docking method by separating the problem into two simpler minimization problems and approximating our energy function by a quadratic equation. These methods provide two new efficient procedures for protein docking computations

3D visualization of HIV virions by cryoelectron tomography.

The structure of the human immunodeficiency virus (HIV) and some of its components have been difficult to study in three-dimensions (3D) primarily because of their intrinsic structural variability. Recent advances in cryoelectron tomography (cryo-ET) have provided a new approach for determining the 3D structures of the intact virus, the HIV capsid, and the envelope glycoproteins located on the viral surface. A number of cryo-ET procedures related to specimen preservation, data collection, and image processing are presented in this chapter. The techniques described herein are well suited for determining the ultrastructure of bacterial and viral pathogens and their associated molecular machines in situ at nanometer resolution

The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle.

Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla. The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate. The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source. The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). Here, we report two high-resolution PduL crystal structures with bound substrates. The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. Accordingly, PduL and Pta exemplify functional, but not structural, convergent evolution. The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen

Single crystal x-ray diffraction studies on small, medium and large molecules

Chapter 1. Production of crystals for diffraction analysis would be assisted by the devising of a set of rules which, given molecular formula, could predict crystal formation conditions. By studying trends in structural properties of a group of closely related simple molecules, deductions could be drawn which could then be applied more generally. Chalcone derivatives with minor substituent differences were recrystallised. X-ray diffraction data collected and the structures solved and refined. Additionally, NMR and UV studies were performed, investigating an observed dimerisation reaction. Chapter 2. Discovery of peptide hormones and neurotransmitters has stimulated the study of structure-activity relationships, although the structure of these molecules is often poorly defined. Proctolin, a linear pentapeptide, is a neurotransmitter in insects. Crystallisation was attempted, with the aim of deducing the active conformation structure, thereby assisting in design of small molecule analogues for use as non-cholinergic pesticides. No diffraction was observed from the crystals produced. Chapter 3. Glucosamine 6-phosphate synthase is an N-terminal nucleophile amidotransferase catalysing the first step in the hexosamine pathway, from which all amino-sugar containing macromolecules are derived. Structure determination of each of two subdomains was attempted. In one case, pseudo-symmetry appeared to obstruct structure solution. The symmetry has subsequently been understood and the structure obtained. Crystals of the second domain are rotationally disordered. Chapters 4 and 5. Recent advances in macromolecular crystallographic techniques have facilitated the collection of an increasing number of high quality, atomic resolution data sets. Methods for refinement, previously limited to small molecule structures, have increasing relevance for proteins. Atomic resolution refinements using these evolving protocols have been performed on two small proteins, rubredoxin from Desulfovibrio vulgaris and the protein G immunoglobulin-binding domain. Appropriate treatment of the solvent structure in a protein crystal and the benefit to be gained by using sharpened density maps during refinement were investigated

Crystal structures of Aldose Reductase, C2A domain of Rabphilin3A and tests of new restraints.

In dieser Arbeit wird die hochaufgelöste (0.82 Å) Struktur der Aldose-Reduktase (ein Enzym, welches am polyol Stoffwechselweg beteiligt ist) vorgestellt. Das Ziel der Arbeit war eine sorgfältige Überprüfung des Modells mit Schwerpunkt auf Merkmale, die nur bei hoher Auflösung erkennbar sind. Daten von atomarer Auflösung erlaubten die Modellierung mehrerer verschiedener Konformationen als in früher bestimmten Strukturen, sowie eine Verbesserung der Übereinstimmung von Modell und Daten. Besondere Aufmerksamkeit wurde der Liste der disagreeable restraints gewidmet: einige kamen wahrscheinlich durch falsche restraints zustande, während andere reale Merkmale der Struktur sind, die nur bei hoher Auflösung sichtbar werden. Diese Beobachtungen könnten verwendet werden, um sowohl eine neue restraints library zu erstellen, als auch um die Molekülstruktur und funktion im Detail zu erklären. Das interessanteste Merkmal des Modells ist die Sicherheitsgurtschleife , die in zwei eigenständigen Konformationen sichtbar ist, der allgemeinen geschlossenen und einer zum Teil geöffneten Konformation (welche möglich ist, weil sich das Enzym in ein post-reaktivem Zustand befindet). Die Aufhebung einer Wechselwirkung genügt, um die Freisetzung des Kofaktors zu bewirken und die Sicherheitsgurtschleife zu öffnen. Die sorgfältige Untersuchung verschiedener hochaufgelöster Aldose-Reduktase Strukturen ergab eine Domänenbewegung die "Zangenbewegung" genannt wurde: die oberen und unteren Schleifen nähern sich dabei an oder entfernen sich voneinander, während das β-barrel starr bleibt. Diese Bewegung könnte mit dem post-reaktiven Zustand des Enzyms und der Freisetzung des Kofaktors zusammenhängen.Weiterhin wird die Kristallstruktur der Ca2+-freien C2A-Domäne von Rabphilin3A (einem neuronalen Protein, das am synaptischen Vesikelzyklus beteiligt ist und dessen genaue biologische Funktion noch ungeklärt ist) bei einer Auflösung von 1.92 Å vorgestellt. Die Struktur besitzt eine Typ1-Topologie und ist durch ein achtsträngiges antiparalleles β-Sandwich charakerisiert. Die elektrostatische Oberfläche ist sehr basisch und weist große Ähnlichkeiten zur C2B-Domäne des Rabphilin3A auf. In der C2A-Domäne des Rabphilin3A wurden zwei strukturelle Merkmale, die durch Unterschiede zur Konsensussequenz zustande kommen, gefunden: eine abweichende Position der Kalziumbindungsschleife 1 (CBL1), wodurch eine deutliche räumliche Verlagerung des konservierten, an der Ca2+-Bindung beteiligten Restes Asp413 verursacht wird, und die Anwesenheit des Restes Glu475 (im DED-Motiv), durch welchen eine lokale negative Ladung nahe der Ca2+-Bindungstelle entsteht. Im Vergleich zu anderen C2 Domänen weist die Struktur andere Merkmale auf, was ein Hinweis auf eine neue Funktion dieser Domäne sein könnte.Im letzten Kapitel werden Überprüfungen mittels einer neuen SHELXL Version, in der ein restraint an ADP gelegt wurde, gezeigt. Der neue restraint imitiert eine TLS Verfeinerung und sollte für Daten mittlerer Auflösung (1.5 - 2.0 Å) nützlich sein. Es wurden Vergleiche zwischen Strukturen, welche sowohl mit TLS in REFMAC als auch mit zwei SHELXL Versionen bei verschiedenen Auflösungen verfeinert wurden, angestellt. Die Ergebnisse zeigen, dass die beiden Programme bei mittlerer Auflösung vergleichbare Daten liefen. Der neue restraint ist allerdings eine gute Verbesserung im Vergleich zu der alten SHELXL Version

A Geometric Approach for Deciphering Protein Structure from Cryo-EM Volumes

Electron Cryo-Microscopy or cryo-EM is an area that has received much attention in the recent past. Compared to the traditional methods of X-Ray Crystallography and NMR Spectroscopy, cryo-EM can be used to image much larger complexes, in many different conformations, and under a wide range of biochemical conditions. This is because it does not require the complex to be crystallisable. However, cryo-EM reconstructions are limited to intermediate resolutions, with the state-of-the-art being 3.6A, where secondary structure elements can be visually identified but not individual amino acid residues. This lack of atomic level resolution creates new computational challenges for protein structure identification. In this dissertation, we present a suite of geometric algorithms to address several aspects of protein modeling using cryo-EM density maps. Specifically, we develop novel methods to capture the shape of density volumes as geometric skeletons. We then use these skeletons to find secondary structure elements: SSEs) of a given protein, to identify the correspondence between these SSEs and those predicted from the primary sequence, and to register high-resolution protein structures onto the density volume. In addition, we designed and developed Gorgon, an interactive molecular modeling system, that integrates the above methods with other interactive routines to generate reliable and accurate protein backbone models

Wordom update 2: A user-friendly program for the analysis of molecular structures and conformational ensembles

We present the second update of Wordom, a user-friendly and efficient program for manipulation and analysis of conformational ensembles from molecular simulations. The actual update expands some of the existing modules and adds 21 new modules to the update 1 published in 2011. The new adds can be divided into three sets that: 1) analyze atomic fluctuations and structural communication; 2) explore ion-channel conformational dynamics and ionic translocation; and 3) compute geometrical indices of structural deformation. Set 1 serves to compute correlations of motions, find geometrically stable domains, identify a dynamically invariant core, find changes in domain-domain separation and mutual orientation, perform wavelet analysis of large-scale simulations, process the output of principal component analysis of atomic fluctuations, perform functional mode analysis, infer regions of mechanical rigidity, analyze overall fluctuations, and perform the perturbation response scanning. Set 2 includes modules specific for ion channels, which serve to monitor the pore radius as well as water or ion fluxes, and measure functional collective motions like receptor twisting or tilting angles. Finally, set 3 includes tools to monitor structural deformations by computing angles, perimeter, area, volume, β-sheet curvature, radial distribution function, and center of mass. The ring perception module is also included, helpful to monitor supramolecular self-assemblies. This update places Wordom among the most suitable, complete, user-friendly, and efficient software for the analysis of biomolecular simulations. The source code of Wordom and the relative documentation are available under the GNU general public license at http://wordom.sf.net

Structure of YraM, a protein essential for growth of Haemophilus influenzae

Nontypeable Haemophilus influenzae is an obligate human parasite that often causes middle ear infections in children and exacerbates chronic obstructive pulmonary disorder, the fourth leading cause of death in the United States. There are no effective vaccines available for this strain. The lipoprotein YraM (gene HI1655) was identified as essential for the growth and viability of H. influenzae but its function is unknown. Sequence comparisons showed that YraM is a fusion of two protein modules. We grew crystals of the carboxyl-terminal module of YraM comprising residues 257–573 (YraM-C), phased the diffraction data by the multiwavelength anomalous diffraction technique, and refined the model to a crystallographic R -factor of 0.16 ( R free = 0.19) with data to 1.35 Å resolution. The two-domain structure of YraM-C adopts a fold similar to that observed for the open, unliganded forms of several periplasmic binding proteins (PBPs) involved in bacterial active transport. Sequence alignments of YraM homologues from other Gram-negative species showed that the most conserved residues of YraM-C cluster between the two domains in the location where other PBPs bind their cognate ligand. Modeling of YraM-C into a closed conformation similar to the leucine-bound form of the Leu/Ile/Val-binding protein (LIVBP) shows a putative binding pocket larger than the leucine-binding site in LIVBP. The pocket has both polar and nonpolar surfaces, with the latter located in the same area where a leucine side chain binds to LIVBP. We discuss possible biological functions of YraM considering its predicted location in the outer membrane, a novel place for such a binding protein. Proteins 2008. © 2008 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/60983/1/22033_ftp.pd

Structural determinants of the domain-selectivity of novel inhibitors of human testis angiotensin-converting enzyme

Includes abstract. Includes bibliographical references (leaves 79-93)