The tertiary structure and backbone dynamics of human prolactin

Human prolactin is a 199-residue (23 kDa) protein closely related to growth hormone and placental lactogen with properties and functions resembling both a hormone and a cytokine. As a traditional hormone, prolactin is produced by lactotrophic cells in the pituitary and secreted into the bloodstream where it acts distally to regulate reproduction and promote lactation. Pituitary cells store prolactin in secretory granules organized around large prolactin aggregates, which are produced within the trans layer of the Golgi complex. Extrapituitary prolactin is synthesized by a wide variety of cells but is not stored in secretory granules. Extrapituitary prolactin displays immunomodulatory activities and acts as a growth factor for cancers of the breast, prostate and tissues of the female reproductive system. We have determined the tertiary structure of human prolactin using three-dimensional (3D) and four-dimensional (4D) heteronuclear NMR spectroscopy. As expected, prolactin adopts an "up-up-down-down" four-helical bundle topology and resembles other members of the family of hematopoietic cytokines. Prolactin displays three discrete structural differences from growth hormone: (1) a structured N-terminal loop in contact with the first helix, (2) a missing mini-helix in the loop between the first and second helices, and (3) a shorter loop between the second and third helices lacking the perpendicular mini-helix observed in growth hormone. Residues necessary for functional binding to the prolactin receptor are clustered on the prolactin surface in a position similar to growth hormone. The backbone dynamics of prolactin were investigated by analysis of 15N NMR relaxation phenomena and demonstrated a rigid four-helical bundle with relatively mobile interconnecting loops. Comparison of global macromolecular tumbling at 0.1mM and 1.0mM prolactin revealed reversible oligomerization, which was correlated to dynamic light scattering experiments. The existence of a reversible oligomerization reaction in solution provides insight into previous results describing the in vitro and in vivo aggregation properties of human prolactin.     

Efficient conformational sampling of local side-chain flexibility

Side-chain flexibility of ligand-binding sites needs to be considered in the rational design of novel inhibitors. We have developed a method to generate conformational ensembles that efficiently sample local side-chain flexibility from a single crystal structure. The rotamer-based approach is tested here for the S1' pocket of human collagenase-1 (MMP-1), which is known to undergo conformational changes in multiple side-chains upon binding of certain inhibitors. First, a raw ensemble consisting of a large number of conformers of the S1' pocket was generated using an exhaustive search of rotamer combinations on a template crystal structure. A combination of principal component analysis and fuzzy clustering was then employed to successfully identify a core ensemble consisting of a low number of representatives from the raw ensemble. The core ensemble contained geometrically diverse conformers of stable nature, as indicated in several cases by a relative energy lower than that of the minimised template crystal structure. Through comparisons with X-ray crystallography and NMR structural data we show that the core ensemble occupied a conformational space similar to that observed under experimental conditions. The synthetic inhibitor RS-104966 is known to induce a conformational change in the side-chains of the S1' pocket of MMP-1 and could not be docked in the template crystal structure. However, the experimental binding mode was reproduced successfully using members of the core ensemble as the docking target, establishing the usefulness of the method in drug design.     

Primary structure of the 2-O-methyl-alpha-L-fucose-containing side chain of the pectic polysaccharide,rhamnogalacturonan II

A 2-O-methylfucosyl-containing heptasaccharide was released from red wine rhamnogalacturonan II (RG-II) by acid hydrolysis of the glycosidic linkage of the aceryl acid residue (AceA) and purified to homogeneity by size-exclusion and high-performance anion-exchange chromatographies. The primary structure of the heptasaccharide was determined by glycosyl-residue and glycosyl-linkage composition analyses, ESIMS, and by 1H and 13C NMR spectroscopy. The NMR data indicated that the pyranose ring of the 2,3-linked L-arabinosyl residue is conformationally flexible. The L-Arap residue was confirmed to be alpha-linked by NMR analysis of a tetraglycosyl-glycerol fragment, [alpha-L-Arap-(1-->4)-beta-D-Galp-(1-->2)-alpha-L-AcefA-(1-->3)-beta-L-Rhap-(1-->3)-Gro], generated by Smith degradation of RG-II. Our data together with the results of a previous study,(1) establish that the 2-O-Me Fuc-containing nonasaccharide side chain of wine RG-II has the structure (Api [triple bond] apiose): [see structure]. Data are presented to show that in Arabidopsis RG-II the predominant 2-O-MeFuc-containing side chain is a mono-O-acetylated heptasaccharide that lacks the non-reducing terminal beta-L-Araf and the alpha-L-Rhap residue attached to the O-3 of Arap, both of which are present on the wine nonasaccharide.     

Refined structure of a flexible heptasaccharide using 1H-13C and 1H-1H NMR residual dipolar couplings in concert with NOE and long range scalar coupling constants

The heptasaccharide isolated from the cell wall polysaccharide of Streptococcus mitis J22 serves as an important model for the dynamics and conformation of complex polysaccharides, illustrating the nature of flexibility with rigid epitopes joined by flexible hinges. One-bond C-H residual dipolar couplings (¹D_CH) and long-range H-H residual dipolar couplings (ⁿD_HH) were measured for the heptasaccharide in a cetylpyridinium chloride/hexanol/brine lamellar liquid crystal medium. A method is proposed to determine the ⁿD_HH in natural abundance based on a ¹³C resolved ¹H TOCSY pulse sequence previously published to determine the homonuclear scalar couplings. Different methods for interpretation of the ¹D_CH and the ⁿD_HH residual dipolar coupling data obtained were compared and combined with the NOE and long-range H,C and C,C scalar couplings available for this heptasaccharide. A flexible model of the heptasaccharide was determined in which two structurally well-defined regions involving four and two sugar residues, respectively are joined by a flexible hinge which involves two 16 glycosidic linkages.     

Role of conformational fluctuations in the enzymatic reaction of HIV-1 protease

The emergence of compensatory drug-resistant mutations in HIV-1 protease challenges the common view of the reaction mechanism of this enzyme. Here, we address this issue by performing classical and ab initio molecular dynamics simulations (MD) on a complex between the enzyme and a peptide substrate. The classical MD calculation reveals large-scale protein motions involving the flaps and the cantilever. These motions modulate the conformational properties of the substrate at the cleavage site. The ab initio calculations show in turn that substrate motion modulates the activation free energy barrier of the enzymatic reaction dramatically. Thus, the catalytic power of the enzyme does not arise from the presence of a pre-organized active site but from the protein mechanical fluctuations. The implications of this finding for the emergence of drug-resistance are discussed.     

Behavioral seasonality in Mahale chimpanzees

To analyze how the chimpanzees in the Mahale Mountains National Park, Tanzania, change their grouping pattern, activity budget, travel speed, and travel distance within an annual cycle, I divided 1-year data into four periods. The Mahale chimpanzees have the behavioral flexibility to adapt to various climates and exhibited at least three behavioral seasons. In the early wet season, chimpanzees formed a few, large parties, and spent much time feeding on insects and animal meat. In the early and late dry seasons, chimpanzees maintained party sizes as large as in the early wet season, and traveled distances similar to the early wet season, but spent the most time feeding and traveling within the year. By contrast, in the late wet season chimpanzee parties broke up into more numerous, small groups, and traveled slowly over shorter distances. Although time spent feeding and traveling were the same as that in the early wet season, time spent feeding on terrestrial herbaceous vegetation (THV) was the highest in the year. The results suggest that chimpanzees travel longer, faster, and farther in seasons when they form large parties.     

Molecular dynamics study of a hyperthermophilic and a mesophilic rubredoxin

In recent years, increased interest in the origin of protein thermal stability has gained attention both for its possible role in understanding the forces governing the folding of a protein and for the design of new highly stable engineered biocatalysts. To study the origin of thermostability, we have performed molecular dynamics simulations of two rubredoxins, from the mesophile Clostridium pasteurianum and from the hyperthermophile Pyrococcus furiosus. The simulations were carried out at two temperatures, 300 and 373 K, for each molecule. The length of the simulations was within the range of 6-7.2 ns. The rubredoxin from the hyperthermophilic organism was more flexible than its mesophilic counterpart at both temperatures; however, the overall flexibility of both molecules at their optimal growth temperature was the same, despite 59% sequence homology. The conformational space sampled by both molecules was larger at 300 K than at 373 K. The essential dynamics analysis showed that the principal overall motions of the two molecules are significantly different. On the contrary, each molecule showed similar directions of motion at both temperatures.     

Fast and accurate side-chain topology and energy refinement (FASTER) as a new method for protein structure optimization

We have developed an original method for global optimization of protein side-chain conformations, called the Fast and Accurate Side-Chain Topology and Energy Refinement (FASTER) method. The method operates by systematically overcoming local minima of increasing order. Comparison of the FASTER results with those of the dead-end elimination (DEE) algorithm showed that both methods produce nearly identical results, but the FASTER algorithm is 100-1000 times faster than the DEE method and scales in a stable and favorable way as a function of protein size. We also show that low-order local minima may be almost as accurate as the global minimum when evaluated against experimentally determined structures. In addition, the new algorithm provides significant information about the conformational flexibility of individual side-chains. We observed that strictly rigid side-chains are concentrated mainly in the core of the protein, whereas highly flexible side-chains are found almost exclusively among solvent-oriented residues.